Human Thrombin Research

Carbon monoxide releasing molecule-2 inhibition of snake venom thrombin-like activity: novel biochemical "brake"?

noreply@blogger.com (Unknown) — Mon, 28 Nov 2016 18:38:00 +0000

Abstract

A complication of defibrinogenation therapy with snake venom enzymes such as ancrod is hypofibrinogenemia associated bleeding secondary to no human-derived inhibitor being available to inactivate or diminish the activity of such enzymes. Of interest, ancrod contains a critical histidine residue without which enzymatic activity is inhibited, and carbon monoxide has been demonstrated to inhibit biomolecular function by interacting with histidine moieties in ion channels.

We tested the hypothesis that exposure of three different snake venoms containing serine proteases with thrombin-like activity (which included ancrod) to carbon monoxide derived from carbon monoxide releasing molecule-2 would diminish their effects on plasmatic coagulation as assessed by thrombelastography. In the case of the Malayan pit viper and Eastern diamondback rattlesnake venoms, carbon monoxide diminished the effects of thrombin-like activity.

In contrast, timber rattlesnake venom demonstrated enhancement of "thrombin-generating" activity with simultaneous loss of thrombin-like activity in response to carbon monoxide exposure. These findings may serve as the rational basis for not just continuing to investigate the potential of snake venom enzymes as clinical defibrinogenating agents, but to also to assess the potential to stop such agents from becoming a catalytic "runaway train" by judicious application of a biochemical "brake" such as carbon monoxide.

PMID:: 27787696
DOI:: 10.1007/s11239-016-1442-4

Thrombin Activity Associated with Neuronal Damage during Acute Focal Ischemia

noreply@blogger.com (Unknown) — Mon, 22 Oct 2012 18:38:00 +0000

Mechanisms of ischemic neuronal and vascular injury remain obscure. Here we test the hypothesis that thrombin, a blood-borne coagulation factor, contributes to neurovascular injury during acute focal ischemia. Stroke was induced in adult Sprague Dawley rats by occluding the middle cerebral artery. Intra-arterial thrombin infusion during ischemia significantly increased vascular disruption and cellular injury. Intravenous infusion of argatroban, a direct thrombin inhibitor, alleviated neurovascular injury. Immunostaining showed thrombin on neurons in the ischemic core.

Using an activatable cell-penetrating peptide engineered to detect thrombin activity, we discovered that thrombin proteolytic activity was specifically associated with neuronal damage during ischemia. Protease activated receptor-1, the presumptive thrombin receptor, appeared to mediate ischemic neurovascular injury. Furthermore, rats receiving thrombin during ischemia showed cognitive deficit, whereas rats receiving argatroban retained intact learning and memory. These results suggest a potential role for thrombin contributing to neurovascular injury and several potential avenues for neuroprotection.

The Journal of Neuroscience, 30 May 2012, 32(22): 7622-7631; doi: 10.1523/JNEUROSCI.0369-12.2012

PubMed citation
Articles by Chen, B.
Articles by Lyden, P. D.

DNA–enzyme conjugate with a weak inhibitor that can specifically detect thrombin in a homogeneous medium

noreply@blogger.com (Unknown) — Mon, 09 May 2011 20:48:00 +0000

Abstract
We present the DNA-assisted control of enzymatic activity for the detection of a target protein using a new type of DNA–enzyme conjugate. The conjugate is composed of an enzyme inhibitor to regulate enzyme activity and a DNA aptamer to be responsive toward the analyte protein. Glutathione S-transferase (GST) and thrombin were selected as a model enzyme and an analyte protein. A hexahistidine tag was genetically attached to the C terminus of the GST, and the 5′ end of an oligonucleotide was conjugated with nitrilotriacetic acid (NTA) for the site-specific conjugation of the DNA with the GST based on a Ni2+ complex interaction. We found that fluorescein acted as a weak inhibitor of GST and succeeded in the regulation of GST activity by increasing the local concentration of the weak inhibitor by the hybridization of a 3′-end fluorescein-modified DNA. The catalytic activity of the DNA aptamer–enzyme conjugate showed a dose-dependent response to thrombin, indicating that the GST activity was clearly recovered by the binding of the DNA aptamer to thrombin. The current system enables the sensitive and specific detection of thrombin simply by measuring the enzymatic activity in a homogeneous medium.

Analytical Biochemistry
Volume 414, Issue 1, 1 July 2011, Pages 103-108

Researchers invent inkjet that prints out living skin

noreply@blogger.com (Unknown) — Mon, 15 Nov 2010 19:54:00 +0000

If you’ve ever seen the lesser-known Sam Raimi movie Darkman, you probably remember that the plot involved the main character, Dr. Westlake, trying to figure out a way to “print” liquid skin to help burn victims. Westlake never did figure out how to keep the synthetic skin from destabilizing past the 98 minute mark, but luckily, Wake Forest Instititute for Regenerative Medicine researchers seem to have mastered it, showing off their amazing skin printer that uses living cells instead of ink.

As the researcher note, “any loss of full-thickness skin of more than 4 cm in diameter will not heal by itself.” Enter their device, which allows a modified inkjet printer to produce reams of fresh skin which can be used to patch up victims of skin trauma. They’ve already tested it on mice, with extremely positive results.

How does the printer work? It has two heads: one dispenses skin cells mixed with a blood coagulent and type I collagen, and the other pumps out thrombin, which is another coagulate. Sprayed together, these chemicals create a reaction and form fibrin, which again helps to clot blood. On top of that, the printer then adds a layer of outer surface skin. Voila!

There’s still testing to be done — the next stage is on pigs, then human trials — but so far, this looks promising… if not to replace burned off skin, then at least to print out some life-like Halloween masks.

Von Willebrand factor and thrombin activation in children with newly diagnosed acute lymphoblastic leukemia: An impact of peripheral blasts†

noreply@blogger.com (Unknown) — Wed, 15 Sep 2010 19:25:00 +0000

Abstract
Background
The pathogenesis and the impact of therapy on thrombin activation in children with acute lymphoblastic leukemia (ALL) are unknown. Steroids may contribute to ALL-associated thrombosis. We explored the hemostatic effects of methylprednisolone monotherapy (MpMT) (32 mg/m2/day IV × 3 days) in children with newly diagnosed ALL.

Methods
Children (>1 to ≤18 years of age) enrolled on DFCI ALL05-01 protocol (n = 30; mean age 6.3 years), without prior steroid therapy, were eligible for study. Overnight fasting pre- and post-MpMT samples were analyzed for coagulation factors [FVIII:C, von Willebrand factor antigen (vWF:Ag) and fibrinogen] and parameters of thrombin generation [prothrombin fragments 1.2 (F1.2), thrombin–antithrombin complex (TAT), and D-dimer].

Results
At diagnosis F1.2 (1.5 nmol/L), TAT (10.9 µg/L), and D-dimers (2,766 ng/ml) levels were increased indicating endogenous thrombin activation. Patients with peripheral blasts (n = 17) had higher levels of vWF:Ag (1.89 vs. 1.14 P = 0.001), TAT (15.39 vs. 5.02 P = 0.038), and D-dimer (3,640 vs. 1,623 P = 0.019) compared to those without peripheral blasts. Following MpMT the blast count decreased significantly from 24% to 3.5% (P < 0.001) with reduction in level of vWF:Ag (1.5, P < 0.01), TAT (8.9, P = 0.42), and D-dimer (P = 0.018) despite 30% increase in FVIII:C levels (P = 0.005). However, patients without peripheral blasts had no significant change in vWF:Ag levels (1.14 vs. 1.25; P = 0.142) and had an increase in thrombin generation parameters.

Conclusions
We postulate that peripheral blasts through endothelial activation stimulate vWF:Ag production/secretion causing coagulation activation. Methylprednisolone therapy reduces the blast count and indirectly suppresses the coagulation activation. Future studies are required to confirm these findings. Pediatr Blood Cancer 2010;54:963–969

Uma Athale MD, MSc1,2,*, Albert Moghrabi MD3, Trishana Nayiager BScH, CCRP2, Yves-Line Delva RN3, Lehana Thabane PhD4,5,6, Anthony K.C. Chan MBBS1,2

Pediatric Blood & Cancer
Volume 54, Issue 7, pages 963–969, 1 July 2010

Bone marrow-derived progenitor cells prevent thrombin-induced increase in lung vascular permeability

noreply@blogger.com (Unknown) — Thu, 28 Jan 2010 18:36:00 +0000

Am J Physiol Lung Cell Mol Physiol 298: L36-L44, 2010

Since thrombin activation of endothelial cells (ECs) is well-known to increase endothelial permeability by disassembly of adherens junctions (AJs) and actinomyosin contractility mechanism involving myosin light chain (MLC) phosphorylation, we investigated the effects of bone marrow-derived progenitor cells (BMPCs) on the thrombin-induced endothelial permeability response.

We observed that addition of BMPCs to endothelial monolayers at a fixed ratio prevented the thrombin-induced decrease in transendothelial electrical resistance, a measure of AJ integrity, and increased mouse pulmonary microvessel filtration coefficient, a measure of transvascular liquid permeability. The barrier protection was coupled to increased vascular endothelial cadherin expression and increased Cdc42 activity in ECs.

Using small interfering RNA (siRNA) to deplete Cdc42 in ECs, we demonstrated a key role of Cdc42 in signaling the BMPC-induced endothelial barrier protection. Endothelial integrity induced by BMPCs was also secondary to inhibition of MLC phosphorylation in ECs. Thus BMPCs interacting with ECs prevent thrombin-induced endothelial hyperpermeability by a mechanism involving AJ barrier annealing, inhibition of MLC phosphorylation, and activation of Cdc42.

Yidan D. Zhao,* Hiroshi Ohkawara,* Stephen M. Vogel, Asrar B. Malik, and You-Yang Zhao
Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois

Thrombin

noreply@blogger.com (Unknown) — Wed, 18 Nov 2009 22:11:00 +0000

Thrombin Applications
Production of fibrin clot in plasma:
Typicallty one to two units of Thrombin will clot one mL of plasma.

Cleavage of Fusion Proteins:
Thrombin can be used for the cleavage of many peptides at the Thrombin recognition site using concentrations of 0.5 NIH units thrombin per one nanomole polypeptide in 20 microliters of 50 mM ammonium bicarbonate, pH 8.0.

Thrombin cleavage of fusion proteins can be carried out at a Thrombin to fusion protein ratio of 1:500.
Fusion proteins may be cleaved in Thrombin cleavage buffer consisting of 50 mM Tris, pH 8.0, 150 mM NaCl, 2.5 mM CaCl2 and 0.1% 2-mercaptoethanol. 2 mg of fusion protein was incubated with 4 µg of thrombin for 20 minutes at RT in the cleavage buffer.

Several conventions are used in Thrombin literature:
1 IOWA unit= 0.83 NIH unit
1 WHO unit = 0.56 NIH unit
1 NIH unit = 0.324 +/- 0.073 µg
1 NIH unit = 1 USP unit

Thrombin (human and bovine) will catalyze the hydrolysis of several peptide p-nitroanilides, tosyl-arg-nitrobenzyl ester, and a thiobenzyl ester synthetic substrates

thrombin references

1.Enzyme Nomenclature: EC 3.4.21.5
2.Chang, J.Y., Eur. J. Biochem., 151, 217?224 (1985).
3.The Plasma Proteins, 2nd ed., 2, Putnam, F. W., ed, p. 148.
4.Machovich, R., The Thrombin, 1, 63-66 (1984)
5.Machovich, R., The Thrombin, 1, 111 (1984)
6.Prasad, S., J. Biol. Chem. 279, 10103-10108 (2004)
7.Kisiel, W., Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin. J. Clin. Invest. 64, 761-769, (1979)
8.The Plasma Proteins, 2nd ed., 2, Putnam, F. W., ed: Table 2. See also: The Enzyme Explorer: Plasma and Blood Protein Resource
9.Qian, W.J., et al., J. Proteome Res., 4, 2070-2080 (2005).
10.Nilsson, B., et al., Arch. Biochem. Biophys., 224, 127-133 (1983)
11.Boyer, P.D., The Enzymes, Academic Press (New York), 3rd ed., Vol. III, p. 277-321 (1971).
12.Expasy/SwissProt: P00743
13.Boissel, J.P., et al., J. Biol. Chem., 259, 5691-5697 1984).
14.Righetti, P.G., and Tudor, G., Isoelectric points and molecular weights of proteins, a new table. Journal of Chromatography, 220, 115-194 (1981).
15.Butkowski, R.J. et al., J. Biol. Chem., 252, 4942 (1977).
16.Winzor, D. J. and Scheraga, H. A., Arch. Biochem. Biophys. 104, 202-207 (1964)
17.Human Blood Coagulation, Haemostasis and Thrombosis, 2nd ed., R. Biggs, ed., p. 722 (1976).
18.The Handbook of Synthetic Substrates, Hemker, H. C., Martinus Nijhoff publisher (1983).
19.Lottenberg, R., et al., Assay of Coagulation Proteases Using Peptide Chromogenic and Fluorogenic Substrates. Meth. Enzymol., 80-C, 341-361 (1981).
20.Chang, Y., Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate. Eur. J. Biochem., 151(2), 217-224 (1985).
21.Hakes, D.J. and Dixon, J.E., Anal. Biochem., 202, 293 (1992).
22.Gaun, KL and Dixon, JE,, Anal. Biochem., 192, 262, 1991
23.De Cristofaro, R. and De Candia, E., J. Thromb. Thrombolysis, 15, 151-163 (2003)
24.Sherwood, J.A., Mol. Biochem. Parisitol., 40, 173-181 (1990)
25.Berg, D.T., et al., Science, 273, 1389-1391 (1996)
26.Lundblad, R.L. et al., Methods Enzymol., 45, 156 (1976)
27.Matsuoka, S., et al., JP. J. Pharmacol., 51, 455-463 (1989)
28.Wimen, B., Meth. Enzymol., 80, 395-408 (1981)
29.Magnusson, S. The Enzymes, 3rd ed., III, pp. 277-321, Boyer, P.D., ed., Academic Press (1971)

Thrombin generation in trauma patients.

noreply@blogger.com (Unknown) — Thu, 20 Aug 2009 15:41:00 +0000

BACKGROUND: Trauma patients are at risk of developing an acute coagulopathy of trauma (ACT) related to tissue injury, shock, and hemodilution. ACT is incompletely understood, but is similar to disseminated intravascular coagulation (DIC) and is associated with poor outcome.

STUDY DESIGN AND METHODS: Thrombin generation assays were used to evaluate plasma hemostasis in 42 trauma patients, 25 normal subjects, and 45 patients on warfarin and in laboratory-prepared factor reduced plasma. RESULTS: Prolonged prothrombin time (PT), more than 18 seconds, or an international normalized ratio of greater than 1.5 was present in 15 trauma patients indicating possible ACT. Native thrombin generation (no activator added, contact activation blocked) showed that Trauma with ACT patients had lag times 68% shorter and peak thrombin generation threefold higher than normal patients indicating the presence of circulating procoagulants capable of initiating coagulation systemically. Trauma patients had lower platelet counts and fibrinogen and Factor (F)II levels putting them at increased risk of bleeding. In laboratory-prepared isolated factor-reduced samples and in patients with vitamin K-dependent factor deficiency due to warfarin, thrombin generation decreased in direct proportion to FII levels. In contrast, in diluted plasma and in trauma patients with reduced factor levels, thrombin generation was increased and associated with slower inhibition of thrombin generation (prolonged termination time) and decreased antithrombin levels (43% of normal in Trauma with ACT).

CONCLUSIONS: Thrombin generation studies indicate that Trauma with ACT patients show dysregulated hemostasis characterized by excessive non-wound-related thrombin generation due to a combination of circulating procoagulants capable of activating coagulation systemically and reduced inhibitor levels allowing systemic thrombin generation to continue once started.

Dunbar NM, Chandler WL.
From the Department of Laboratory Medicine, University of Washington, Seattle, Washington

Assessment of thrombin generation: useful or hype?

noreply@blogger.com (Unknown) — Wed, 20 May 2009 20:07:00 +0000

School of Human Life Sciences, University of Tasmania, Tasmania, Australia. Murray.

Thrombin plays an important role in hemostasis through its multiple functions across blood coagulation, platelet activation, and fibrinolysis. The measurement of thrombin generation is therefore viewed as a potentially useful test that could be applied to the screening, monitoring, and/or diagnosis of hemostatic abnormalities. Indeed, advances in thrombin generation assays have created significant interest and debate as to whether they may provide a more physiologically relevant testing system than do traditional coagulation tests.

A variety of thrombin generation assays, including commercially available systems, have been investigated for their correlation with hypocoagulable and hypercoagulable states. Although there is an extensive body of literature that has investigated the application of thrombin generation assays, some limitations remain. These include poor standardization of reagents and methods and a lack of large prospective studies that demonstrate clear relationships between thrombin generation with bleeding and thrombosis phenotypes, as well as with monitoring anticoagulation.

Whether thrombin generation assays become more "useful" than "hype" will require well-designed, large, prospective multicenter trials using standardized methods

What is Thrombin

noreply@blogger.com (Unknown) — Fri, 09 Jan 2009 21:29:00 +0000

THROMBIN(activated Factor II [IIa]) is a coagulation protein that has many effects in the coagulation cascade. It is a serine protease (EC 3.4.21.5) that converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions.

Activation of prothrombin is crucial in physiological and pathological coagulation. Various rare diseases involving prothrombin have been described (e.g., hypoprothrombinemia). Anti-thrombin antibodies in autoimmune disease may be a factor in the formation of the lupus anticoagulant also known as ( antiphospholipid syndrome ).

In addition to its activity in the coagulation cascades, thrombin also promotes platelet activation, via activation of protease-activated receptors on the platelet.

Thrombin, a potent vasoconstrictor and mitogen, is implicated as a major factor in vasospasm following subarachnoid hemorrhage. Blood from a ruptured cerebral aneurysm clots around a cerebral artery, releasing thrombin. This can induce an acute and prolonged narrowing of the blood vessel, potentially resulting in cerebral ischemia and infarction (stroke).

Due to its high proteolytic specificity, thrombin is a valuable biochemical tool. The thrombin cleavage site (Leu-Val-Pro-Arg-Gly-Ser) is commonly included in linker regions of recombinant fusion protein constructs. Following purification of the fusion protein, thrombin can be used to selectively cleave between the Arginine and Glycine residues of the cleavage site, effectively removing the purification tag from the protein of interest with a high degree of specificity.

References
^ McMillen, S.I. (1984), None of These Diseases (Old Tappan, NJ: Revell)
^ Schmidt A (1872). "Neue Untersuchungen ueber die Fasserstoffesgerinnung". Pflüger's Archiv für die gesamte Physiologie 6: 413–538. doi:10.1007/BF01612263.

Recommended Reading
Further reading
Esmon CT (1995). "Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface". Faseb J 9 (10): 946–55. PMID 7615164.
Lenting PJ, van Mourik JA, Mertens K (1999). "The life cycle of coagulation factor VIII in view of its structure and function". Blood 92 (11): 3983–96. PMID 9834200.
Plow EF, Cierniewski CS, Xiao Z, et al. (2002). "AlphaIIbbeta3 and its antagonism at the new millennium". Thromb. Haemost 86 (1): 34–40. PMID 11487023.
Maragoudakis ME, Tsopanoglou NE, Andriopoulou P (2002). "Mechanism of thrombin-induced angiogenesis". Biochem. Soc. Trans 30 (2): 173–7. doi:10.1042/ (inactive 28 June 2008). PMID 12023846.
Howell DC, Laurent GJ, Chambers RC (2002). "Role of thrombin and its major cellular receptor, protease-activated receptor-1, in pulmonary fibrosis". Biochem. Soc. Trans 30 (2): 211–6. doi:10.1042/ (inactive 28 June 2008). PMID 12023853.
Firth SM, Baxter RC (2003). "Cellular actions of the insulin-like growth factor binding proteins". Endocr. Rev 23 (6): 824–54. doi:10.1210/er.2001-0033. PMID 12466191.
Minami T, Sugiyama A, Wu SQ, et al. (2004). "Thrombin and phenotypic modulation of the endothelium". Arterioscler. Thromb. Vasc. Biol 24 (1): 41–53. doi:10.1161/01.ATV.0000099880.09014.7D. PMID 14551154.
De Cristofaro R, De Candia E (2004). "Thrombin domains: structure, function and interaction with platelet receptors". J. Thromb. Thrombolysis 15 (3): 151–63. doi:10.1023/B:THRO.0000011370.80989.7b. PMID 14739624.
Tsopanoglou NE, Maragoudakis ME (2004). "Role of thrombin in angiogenesis and tumor progression". Semin. Thromb. Hemost 30 (1): 63–9. doi:10.1055/s-2004-822971. PMID 15034798.
Bode W (2007). "Structure and interaction modes of thrombin". Blood Cells Mol. Dis 36 (2): 122–30. doi:10.1016/j.bcmd.2005.12.027. PMID 16480903.
Wolberg AS (2007). "Thrombin generation and fibrin clot structure". Blood Rev 21 (3): 131–42. doi:10.1016/j.blre.2006.11.001. PMID 17208341.
Degen S: Prothrombin. In: High K, Roberts H, eds. Molecular Basis of Thrombosis and Hemostasis. New York, NY: Marcel Dekker; 1995:75.
[show]v • d • eProteins: coagulation

Vascular smooth muscle cells for use in vascular tissue engineering obtained by endothelial-to-mesenchymal transdifferentiation (EnMT) on collagen...

noreply@blogger.com (Unknown) — Thu, 17 Jul 2008 20:50:00 +0000

The discovery of the endothelial progenitor cell (EPC) has led to an intensive research effort into progenitor cell-based tissue engineering of (small-diameter) blood vessels. Herein, EPC are differentiated to vascular endothelial cells and serve as the inner lining of bioartificial vessels. As yet, a reliable source of vascular smooth muscle progenitor cells has not been identified. Currently, smooth muscle cells (SMC) are obtained from vascular tissue biopsies and introduce new vascular pathologies to the patient. However, since SMC are mesenchymal cells, endothelial-to-mesenchymal transdifferentiation (EnMT) may be a novel source of SMC. Here we describe the differentiation of smooth muscle-like cells through EnMT. Human umbilical cord endothelial cells (HUVEC) were cultured either under conditions favoring endothelial cell growth or under conditions favoring mesenchymal differentiation (TGF-β and PDGF-BB). Expression of smooth muscle protein 22 and -smooth muscle actin was induced in HUVEC cultured in mesenchymal differentiation media, whereas hardly any expression of these markers was found on genuine HUVEC. Transdifferentiated endothelial cells lost the ability to prevent thrombin formation in an in vitro coagulation assay, had increased migratory capacity towards PDGF-BB and gained contractile behavior similar to genuine vascular smooth muscle cells. Furthermore, we showed that EnMT could be induced in three-dimensional (3D) collagen sponges. In conclusion, we show that HUVEC can efficiently transdifferentiate into smooth muscle-like cells through endothelial-to-mesenchymal transdifferentiation. Therefore, EnMT might be used in future progenitor cell-based vascular tissue engineering approaches to obtain vascular smooth muscle cells, and circumvent a number of limitations encountered in current vascular tissue engineering strategies.

ARTICLE

Progesterone metabolites rapidly stimulate calcium influx in human platelets by a src-dependent pathway

noreply@blogger.com (Unknown) — Mon, 07 Jul 2008 18:30:00 +0000

The effects of several steroids and their metabolites were examined for their ability to rapidly alter intracellular free calcium ([Ca2+]i) in the anucleate human platelet. Earlier studies suggested that steroids had direct and rapid non-genomic effects to alter platelet physiology. The rationale for performing this study was to investigate the signal transduction events being activated by steroids. Super-physiologic concentrations (1.0–10.0 μM) of β-estradiol and several estradiol metabolites and analogs potentiated (approximately twofold) the action of thrombin to elevate [Ca2+]i in platelets, whereas 10.0 μM progesterone inhibited the action of human thrombin by 10–15%. Progesterone and β-estradiol by themselves did not affect [Ca2+]i. Progesterone metabolites can achieve high blood concentrations. Some progesterone metabolites, particularly those in the β-conformation, were potent stimulators of Ca2+ influx and intracellular Ca2+ mobilization in platelets. They activated phospholipase C because their ability to increase [Ca2+]i was inhibited by the phospholipase C inhibitor U-73122. The ability of pregnanediol and collagen to increase [Ca2+]i was inhibited by the src tyrosine kinase inhibitor PP1, whereas the actions of thrombin and thapsigargin to increase [Ca2+]i were not affected by PP1. The effects of progesterone metabolites to increase [Ca2+]i were observed with concentrations as low as 0.1 μM. Pregnanolone synergized with thrombin to increase [Ca2+]i. It is hypothesized that human platelets possess receptors for progesterone metabolites. These receptors when stimulated will activate platelets by causing a rapid increase in [Ca2+]i. Pregnanolone, isopregnanediol and pregnanediol were the most effective stimulators of this newly identified src-dependent signal transduction system in platelets. Progesterone metabolites may regulate platelet aggregation and hence thrombosis in vivo.

ARTICLE

Thrombin regulates CD40 expression in microglial cells

noreply@blogger.com (Unknown) — Tue, 01 Jul 2008 17:46:00 +0000

Microglial cells are the innate immune cells of the central nervous system and quickly respond to injury by proliferation, cytokine release, and increased cell surface antigen expression. Thrombin is a multifunctional serine proteinase, which has the capability to activate microglial cells. Here, we report that pharmaceutical-grade thrombin dose-dependently increases the expression of CD40 in N9 microglial cells. This effect is blocked by a thrombin inhibitor, mimicked by thrombin receptor-activating peptide and modified by mitogen-activated protein kinase pathway inhibitors. Thrombin-induced CD40 regulation might play a role in diseases with breakdown of the blood-brain barrier such as multiple sclerosis or stroke.

ARTICLE

A Phase 3, Randomized, Double-Blind Comparative Study of the Efficacy and Safety of Topical Recombinant Human Thrombin and Bovine Thrombin in Surgical

noreply@blogger.com (Unknown) — Fri, 20 Jun 2008 20:49:00 +0000

Background
Plasma-derived bovine thrombin is used as a topical agent to improve surgical hemostasis, but development of antibodies to bovine hemostatic proteins has been associated with increased bleeding and thrombotic complications. Recombinant human thrombin could reduce the risk of these complications.

Study Design
The objective of this randomized, double-blind, comparative trial was to compare the efficacy, safety, and antigenicity of recombinant human thrombin (rhThrombin) and bovine thrombin as adjuncts to hemostasis in liver resection, spine, peripheral arterial bypass, and dialysis access surgery. Blinded study drug was applied topically to bleeding sites with an absorbable gelatin sponge. The primary efficacy end point was time to hemostasis, summarized as the incidence of hemostasis within 10 minutes. Safety analyses were conducted for 1 month after operation, and the development of antibodies to rhThrombin or to the bovine product was evaluated.

Results
Four hundred one patients completed this trial. Hemostasis was achieved at the time-to-hemostasis evaluation site within 10 minutes in 95% of patients in each treatment group. Overall complications, including operative mortality, adverse events, and laboratory abnormalities, were similar between groups. Forty-three (21.5%) patients receiving bovine thrombin developed antibodies to the product; three patients (1.5%; p < 0.0001) in the rhThrombin group developed antibodies to rhThrombin. None of the three patients who developed antirhThrombin antibodies had abnormal coagulation laboratory results or bleeding, thromboembolic, or hypersensitivity events.

Conclusions
Results of this trial suggest that rhThrombin has comparable efficacy, a similar safety profile, and is considerably less immunogenic than bovine thrombin when used for surgical hemostasis.

FDA Approves Human Thrombin for Topical Use in Surgery

noreply@blogger.com (Unknown) — Fri, 20 Jun 2008 20:44:00 +0000

The U.S. Food and Drug Administration approved Evithrom (Human Thrombin), a blood-clotting protein used to help control bleeding during surgery.

Evithrom is the first human thrombin approved since 1954 and is the only product currently licensed. It is derived from human plasma obtained from carefully screened and tested U.S. donors and has undergone steps to further reduce the risk for transfusion-transmitted diseases.

Evithrom is indicated as an aid to stop oozing and minor bleeding from capillaries and small veins and when control of bleeding by standard surgical techniques is ineffective or impractical. The product is applied to the surface of bleeding tissue and may be used in conjunction with an absorbable gelatin sponge. Evithrom must not be injected into blood vessels, which would result in serious clinical complications and may even be fatal.

"The approval of Evithrom offers an important additional option for surgeons and their patients to help control surgical bleeding," said Jesse L. Goodman, M.D., M.P.H., director of FDA's Center for Biologics Evaluation and Research. "Surgeons will now be able to choose between human thrombin and thrombin derived from cattle plasma."

In a clinical trial involving several hundred subjects, Evithrom was found comparable to cattle-derived thrombin in both safety and effectiveness.

Evithrom is manufactured by Omrix Biopharmaceuticals, Ltd., Ramat Gan, Israel, and will be distributed by Johnson & Johnson Wound Management, a division of Ethicon, Inc., Somerville, N.J.

Role of the Endothelium in the Vascular Effects of the Thrombin Receptor (Protease-Activated Receptor Type 1) in Humans

noreply@blogger.com (Unknown) — Mon, 02 Jun 2008 22:07:00 +0000

Objectives
The purpose of this study was to determine the role of the endothelium in the vascular actions of protease-activated receptor type 1(PAR-1) activation in vivo in man.

Background
Thrombin is central to the pathophysiology of atherothrombosis. Its cellular actions are mediated via PAR-1. Protease-activated receptor type 1 activation causes arterial vasodilation, venoconstriction, platelet activation, and tissue-type plasminogen activator release in man.

Methods
Dorsal hand vein diameter was measured in 6 healthy volunteers before and after endothelial denudation. Forearm arterial blood flow, plasma fibrinolytic factors, and platelet activation were measured in 24 healthy volunteers during venous occlusion plethysmography. The effects of inhibition of prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor on PAR-1 responses were assessed during coadministration of aspirin, the “NO clamp” (L-NG-monomethyl arginine and sodium nitroprusside), and tetraethylammonium ion, respectively.

Results
Endothelial denudation did not affect PAR-1–evoked venoconstriction (SFLLRN; 0.05 to 15 nmol/min). Although aspirin had no effect, SFLLRN-induced vasodilation (5 to 50 nmol/min) was attenuated by the NO clamp (p < 0.0001) and tetraethylammonium ion (p < 0.05) and abolished by their combination (p < 0.01). The NO clamp augmented SFLLRN-induced tissue-type plasminogen activator and plasminogen activator inhibitor type 1 antigen (p < 0.0001) release, but tetraethylammonium ion and aspirin had no effect. SFLLRN-induced platelet activation was unaffected by NO or prostacyclin inhibition.

Conclusions
Acting via PAR-1, thrombin causes contrasting effects in the human vasculature and has a major interaction with the endothelium. This highlights the critical importance of endothelial function during acute arterial injury and intravascular thrombosis, as occurs in cardiovascular events including myocardial infarction and stroke.

Role of the Endothelium in the Vascular Effects of the Thrombin Receptor (Protease-Activated Receptor Type 1) in Humans

Thrombin is central to the pathophysiology of atherothrombosis and exerts its vascular effects via protease activated receptor type 1 (PAR-1). We report the contrasting role of the endothelium in PAR-1 activation in vivo in man, where it mediates arterial PAR-1–induced vasodilation and tissue plasminogen activator release but does not provide a major contribution to venous tone or plasminogen activator inhibitor type 1 release. Our findings provide evidence of a major interaction between the endothelium and thrombin in vivo. This highlights the critical importance of endothelial function during acute arterial injury and thrombosis, as occurs in acute coronary syndromes.

Ingibjörg J. Gu mundsdóttir, Ninian N. Lang, Nicholas A. Boon, Christopher A. Ludlam, David J. Webb, Keith A. Fox, David E. Newby

Role of thrombin in interleukin-5 expression from basophils.

noreply@blogger.com (Unknown) — Wed, 06 Feb 2008 17:07:00 +0000

Interleukin-5 (IL-5) plays a key role in the pathogenesis of bronchial asthma. Thrombin is a procoagulant factor that has been also reported to participate in the inflammatory response by stimulating the secretion of cytokines . Interaction of inflammatory cells with airway epithelial cells may also promote the secretion of cytokines. However, the role of Thrombin and cell-to-cell interaction in pathogenesis of allergic inflammation is unclear. In this study, we evaluated the role of thrombin and cell-to-cell interaction in the secretion of IL-5 from basophils. The human basophil cell line KU-812 was used in the assays. Thrombin and co-culture with alveolar epithelial cells significantly stimulated the secretion of IL-5 from KU-812 cells as compared to controls. Secretion of IL-5 was synergistically stimulated when KU-812 cells were incubated in the presence of both thrombin and alveolar epithelial cells. Co-culture of KU-812 cells with epithelial cells significantly increased the expression of tissue factor, an activator of coagulation activation, in a cell dose-dependent manner. Secretion of IL-5 from KU-812 basophils co-cultured with epithelial cells was significantly inhibited by LY294002, an inhibitor of phosphatidylinositol 3-kinase. These results suggest that thrombin and cell interaction with lung epithelial cells may augment the inflammatory response in allergic diseases by stimulating the secretion of IL-5 from basophils .

Yamaguchi A, Gabazza EC, Takei Y, Yano Y, Fujimoto H, D'Alessandro-Gabazza CN, Murakami E, Kobayashi T, Takagi T, Maruyama J, Suzuki K, Taguchi O.
Department of Pulmonary and Critical Care Medicine, Mie University Graduate School of Medicine, Edobashi 2-174, Tsu City, Mie Prefecture, Japan.Biochem Biophys Res Commun. 2008 Jan 18