Calin from Hirudo medicinalis, an inhibitor of platelet adhesion to collagen, prevents platelet-rich thrombosis in hamsters

Interaction between exposed collagen and platelets and/or von Willebrand factor is believed to be one of the initiating events for thrombus formation at sites of damaged endothelium. Interference with this mechanism may provide an anti-thrombotic potential. Calin, a product from the saliva of the leech Hirudo medicinalis, was tested in vitro and for its in vivo activity in a thrombosis model in hamsters. Calin specifically and dose dependently (IC50:6.5 to 13 micrograms/mL) inhibited human platelet aggregation induced by collagen. In addition, specific platelet adhesion onto microtiter wells coated with collagen and detected with a monoclonal antiglycoprotein IIb/IIIa antibody- conjugated with horseradish peroxidase, could be completely prevented with Calin (IC50:22 micrograms/mL). A dose-response curve was constructed in groups of six hamsters in whom a standardized trauma was induced on the femoral vein. Thrombus formation was followed continuously using video recording and processing of the image obtained upon transillumination of the vessel. Intravenous Calin dose- dependently inhibited platelet-rich thrombus formation in this model with an ED50 of 0.07 mg/kg and complete inhibition with 0.2 mg/kg. No effects were seen on coagulation tests or bleeding times, whereas ex vivo aggregation induced by collagen was inhibited dose dependently. Local application of leech saliva, Calin, hirudin, or the combination of the latter two into the bleeding time wound of hamsters resulted in a mild prolongation of the bleeding time (twofold to threefold). A similar experiment in baboons did not cause any prolongation of the bleeding time. This is in sharp contrast with the long-lasting bleeding after a leech bite itself in both species. Calin from the leech Hirudo medicinalis is able, by binding to collagen, to effectively interfere with platelet-collagen interaction, which results in an antithrombotic effect observed in a platelet-rich thrombosis model in hamsters.

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Antithrombotic Effects and Bleeding Time Prolongation with Synthetic Platelet GPIIb/llla Inhibitors in Animal Models of Platelet-Mediated Thrombosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642390 ◽

1994 ◽

Vol 71 (01) ◽

pp. 095-102 ◽

Cited By ~ 43

Author(s):

Désiré Collen ◽

Hua Rong Lu ◽

Jean-Marie Stassen ◽

Ingrid Vreys ◽

Tsunehiro Yasuda ◽

...

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Bolus Injection ◽

Cell Injury ◽

Ex Vivo ◽

Thrombus Formation ◽

Femoral Vein ◽

Thrombotic Occlusion ◽

Antithrombotic Effects

SummaryCyclic Arg-Gly-Asp (RGD) containing synthetic peptides such as L-cysteine, N-(mercaptoacetyl)-D-tyrosyl-L-arginylglycyl-L-a-aspartyl-cyclic (1→5)-sulfide, 5-oxide (G4120) and acetyl-L-cysteinyl-L-asparaginyl-L-prolyl-L-arginyl-glycyl-L-α-aspartyl-[0-methyltyrosyl]-L-arginyl-L-cysteinamide, cyclic 1→9-sulfide (TP9201) bind with high affinity to the platelet GPIIb/IIIa receptor.The relationship between antithrombotic effect, ex vivo platelet aggregation and bleeding time prolongation with both agents was studied in hamsters with a standardized femoral vein endothelial cell injury predisposing to platelet-rich mural thrombosis, and in dogs with a carotid arterial eversion graft inserted in the femoral artery. Intravenous administration of G4120 in hamsters inhibited in vivo thrombus formation with a 50% inhibitory bolus dose (ID50) of approximately 20 μg/kg, ex vivo ADP-induccd platelet aggregation with ID50 of 10 μg/kg, and bolus injection of 1 mg/kg prolonged the bleeding time from 38 ± 9 to 1,100 ± 330 s. Administration of TP9201 in hamsters inhibited in vivo thrombus formation with ID50 of 30 μg/kg, ex vivo platelet aggregation with an ID50 of 50 μg/kg and bolus injection of 1 mg/kg did not prolong the template bleeding time. In the dog eversion graft model, infusion of 100 μg/kg of G4120 over 60 min did not fully inhibit platelet-mediated thrombotic occlusion but was associated with inhibition of ADP-induccd ex vivo platelet aggregation and with prolongation of the template bleeding time from 1.3 ± 0.4 to 12 ± 2 min. Infusion of 300 μg/kg of TP9201 over 60 min completely prevented thrombotic occlusion, inhibited ex vivo platelet aggregation, but was not associated with prolongation of the template bleeding time.TP9201, unlike G4120, inhibits in vivo platelet-mediated thrombus formation without associated prolongation of the template bleeding time.

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Novel 12-LOX Inhibitor ML355 Attenuates Platelet Reactivity and Impairs Thrombus Growth, Stability and Vessel Occlusion In Vivo

Blood ◽

10.1182/blood.v126.23.3442.3442 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3442-3442 ◽

Cited By ~ 1

Author(s):

Reheman Adili ◽

Theodore R Holman ◽

Michael Holinstat

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Platelet Adhesion ◽

Ex Vivo ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Vessel Occlusion ◽

Growth Stability

Abstract Background: Adequate platelet reactivity is required for platelet adhesion and aggregation at the site of vascular injury to maintain hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi, the predominate underlying cause of myocardial infarction and stroke. While current anti-platelet treatments limit platelet function, they often result in an increased risk of bleeding. 12-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated by our lab and others to regulate PAR4 and GPVI-mediated platelet reactivity suggesting a role of 12-LOX in regulation of vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Aims: To determine how 12-LOX regulates thrombus formation in vivo and whether platelet 12-LOX is an effective target for anti-platelet therapeutics, wild-type (WT) or 12-LOX deficient (12-LOX-/-) mice were treated with or without the 12-LOX inhibitor, ML355, and were assessed for inhibitory effects on platelet activation in vitro, ex-vivo and in vivo. Methods: The effect of the novel 12-LOX inhibitor ML355 on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber. In vivo thrombus formation and vessel occlusion in small and large vessels were studied in 12-LOX-/-, WT mice and mice treated with ML355 using intravital microscopy using the FeCl3 injury models. Results: Using in vitro platelet aggregation assays, ML355 dose dependently inhibited thrombin, PAR1-AP, and PAR4-AP-induced aggregation in washed human platelets. Interestingly, the negative regulatory effects of ML355 inhibition of 12-LOX can be overcome by high concentration of thrombin. Additionally, ML355 was able to attenuate ADP-induced platelet aggregation both in platelet-rich-plasma and whole blood. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX-/- mice was impaired in FeCl3-induced mesenteric or carotid artery thrombosis models. Thrombi in 12-LOX-/- mice were unstable and frequently form emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The highly selective 12-LOX inhibitor ML355 inhibits platelets aggregation induced by various platelet agonists and ML355 inhibition of platelet function is not agonist specific. Platelet function at high shear in ex vivo conditions in both mice and human was attenuated in the presence of ML355. Thrombus growth, stability, and vessel occlusion was impaired in mice deficient for 12-LOX. Finally, the highly selective 12-LOX inhibitor ML355 attenuates thrombus formation and prevents vessel occlusion in vivo. Our data strongly indicates 12- LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics. Disclosures No relevant conflicts of interest to declare.

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Antithrombotic Effect of a Recombinant von Willebrand Factor, VCL, on Nitrogen Laser-Induced Thrombus Formation in Guinea Pig Mesenteric Arteries

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653771 ◽

1995 ◽

Vol 73 (02) ◽

pp. 318-323 ◽

Cited By ~ 20

Author(s):

K Azzam ◽

L I Garfinkel ◽

C Bal dit Sollier ◽

M Cisse Thiam ◽

L Drouet

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Ex Vivo ◽

Thrombus Formation ◽

Mesenteric Arteries ◽

Antithrombotic Effects ◽

Von Willebrand ◽

Willebrand Factor

SummaryTo assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded.In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i. v. injection inhibits ristocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time. In vivo, VCL (0.5 to 2 mg/kg) had no effect on the appearance of the thrombi formed but produced dose-dependent inhibition of the mean number of recurrent thrombi (the maximal effect was obtained at 5 min following i. v. injection of the highest dose: 0.8 ± 0.2 thrombi versus 4 ± 0.4 thrombi in controls). The three doses of VCL increased the time in which the first thrombus in a concentration-dependent manner was formed. However, the time to visualize the first thrombus was only prolonged in the higher dose-treated group.These in-vivo studies confirm that VCL induces immediate, potent, and transient antithrombotic effects. Most importantly, this inhibition was achieved without inducing thrombocytopenia nor prolongation of the bleeding time.

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Anti-Human VWF Monoclonal Antibody SZ-123 Prevents Arterial Thrombus Formation by Inhibiting VWF–collagen and VWF-Platelet Interactions in Rhesus Monkeys

Blood ◽

10.1182/blood.v120.21.5194.5194 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5194-5194

Author(s):

Yiming Zhao ◽

Changgeng Ruan

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Bleeding Time ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Thrombus Formation ◽

Significant Prolongation ◽

Arterial Thrombus

Abstract Abstract 5194 Objective: To investigate the in vivo antithrombotic efficacy of an anti-VWF monoclonal antibody SZ-123, and its potential underlying mechanism. Methods and Results: Cyclic flow reductions (CFRs) were measured in the femoral artery of monkeys before and after intravenous administration of SZ-123. Ex vivo VWF binding to collagen, platelet aggregation, platelet count and template bleeding time were performed as measurements of antithrombotic activity. In addition, plasma VWF, SZ-123 levels, and VWF occupancy were measured by ELISA. Administration of 0. 1, 0. 3, and 0. 6 mg/kg SZ-123 resulted in 45. 3%, 78. 2%, and 100% reduction in CFRs, respectively. When 0. 3 and 0. 6 mg/kg SZ-123 were administrated, 100% of VWF was occupied by the antibody. Moreover, 100% ex vivo inhibition of VWF-collagen binding and 60–95% inhibition of platelet aggregation were observed from 15 min to 1h. None of the doses resulted in significant prolongation of bleeding time. In vitro experiment also revealed that SZ-123 not only blocks collagen-VWF A3 interaction but also inhibits indirectly VWF A1 binding to GPIba induced by ristocetin. Conclusions: SZ-123 prevents in vivo arterial thrombus formation under high shear conditions by inhibiting VWF A3–collagen and VWF A1-platelet interactions and does not prolong bleeding time. Disclosures: No relevant conflicts of interest to declare.

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ROLE OF NITRIC OXIDE (NO) IN CAPSAICIN MEDIATED ANTI-PLATELET ACTIVITY IN IN VITRO, IN VIVO, EX-VIVO MODEL OF PLATELET AGGREGATION ASSAY AND ARTERIAL THROMBOSIS IN RAT: POTENTIAL THERAPEUTIC TARGET?

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2018v10i4.22474 ◽

2018 ◽

Vol 10 (4) ◽

pp. 44

Author(s):

Mihir K Patel ◽

Kiranj K. Chaudagar ◽

Anita A. Mehta

Keyword(s):

Platelet Aggregation ◽

Arterial Thrombosis ◽

Ex Vivo ◽

Platelet Activity ◽

Aggregation Assay ◽

Thrombosis Model ◽

Platelet Aggregation Assay

Objective: Although recent advances in the treatment of congestive heart disease, mortality among patients’ remains a questionable remark. Therefore, we evaluated the role of capsaicin on in vitro and ex vivo platelet aggregation induced by Adenosine Di-Phosphate (ADP) as well as in in vivo thrombosis models and role of NO, KATP was also identified in the capsaicin-induced anti-platelet animal model as well as in vivo model of arterial thrombosis.Methods: According to body weight wistar rats were divided into five groups. Group I and Group II was treated with saline and capsaicin (3 mg/kg, i. v), while animals from Group III were treated with N(ω)-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg, i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group IV animals were treated with glibenclamide (10 mg/kg,i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group V was considered as a positive control and administered clopidogrel (30 mg/kg, p. o). Animals were subjected for in vitro, ex-vivo platelet aggregation assay. ADP (30µM) was utilized as an aggregating agent in these experiments. After these assays; animals of each group were subjected for subaqueous tail bleeding time in a rat model and FeCl3-induced arterial thrombosis model in rats.Results: In ADP-induced in vitro platelet aggregation, a significant reduction in % platelet aggregation was observed at 50µM (64.35±4.641) and 100µM (52.72±4.192) concentration of capsaicin as compared to vehicle control (85.82±3.716). Capsaicin (3 mg/kg, i. v) also showed a significant reduction (49.53±4.075) in ex-vivo ADP-induced platelet aggregation as compared to vehicle control (89.38±2.057). In FeCl3 induced arterial thrombosis model, Capsaicin (3 mg/kg, i. v) exhibited an increase in time to occlusion in this rodent model and presence of the L-NAME and glibenclamide had inhibited the activity of capsaicin.Conclusion: In our study, capsaicin (50 µM, 100µM) exhibited potent anti-platelet activity in ADP-induced platelet aggregation, similarly capsaicin exhibited significant anti-platelet action in the ex-vivo study. Moreover, the presence of L-NAME and glibenclamide inhibited the anti-thrombotic and anti-platelet action of capsaicin. Therefore, it was concluded that NO and KATP may be involved in the anti-thrombotic action of capsaicin.

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Abstract 54: ML355 in Prevention of Thrombosis in vivo with Minimal Effects on Hemostasis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.54 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Reheman Adili ◽

Katherine Mast ◽

Michael Holinstat

Keyword(s):

Ex Vivo ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Flow Chamber ◽

Human Platelets ◽

Mass Spectrometry Analysis ◽

Vessel Occlusion ◽

Spectrometry Analysis ◽

Thrombosis Model

12-lipoxygenase (12-LOX) has been demonstrated to regulate platelet function, hemostasis, and thrombosis ex vivo , supporting a key role for 12-LOX in regulation of in vivo thrombosis. While pharmacologically targeting 12-LOX in vivo has been a challenge to date, the recent development of the 12-LOX selective inhibitor, ML355, as an effective antiplatelet therapeutic in vivo was assessed. ML355 potently inhibited thrombin and other agonist-induced platelet aggregation ex vivo in washed human platelets and inhibited downstream oxylipin production of platelet 12-LOX as confirmed by Mass spectrometry analysis. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen was attenuated in human whole blood treated with ML355 to a greater extent compared to aspirin. In vivo , PK assessment of ML355 showed reasonable 12-LOX plasma levels 12 hours following administration of ML355. FeCl 3 -induced injury of the mesenteric arterioles resulted in less stable thrombi in 12-LOX -/- mice and ML355-treated WT mice resulting in impairment of vessel occlusion. Additionally, ML355 dose-dependently inhibited laser-induced thrombus formation in the cremaster arteriole thrombosis model in WT, but not in 12-LOX -/- mice. Importantly, hemostatic plug formation and bleeding following treatment with ML355 were not affected in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. Our data strongly supports 12-LOX as a key determinant of platelet reactivity in vivo and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapeutics.

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Effects of Sodium Nitroprusside on Platelet Adhesion, Subsequent Aggregation and Vasoconstriction

10.1055/s-0039-1682743 ◽

1977 ◽

Author(s):

H.R. Baumgartner

Keyword(s):

Myocardial Infarction ◽

Acute Myocardial Infarction ◽

Sodium Nitroprusside ◽

Platelet Adhesion ◽

Ex Vivo ◽

Balloon Catheter ◽

Strong Inhibitor ◽

Induced Aggregation

Sodium nitroprusside (SNP), a potent vasodilator, has shown beneficial effects in acute myocardial infarction. Since platelets may play an important role in the pathogenesis of myocardial infarction, the effect of SNP on their interaction with rabbit aorta subendothelium was investigated in vivo and under controlled blood flow conditions ex vivo and in vitro.One iliac artery and the abdominal aorta were denuded of endothelium by balloon catheter injury during infusion of glucose, SNP at 6 or 12 μg/kg/min in groups of 12, 6 and 7 rabbits respectively. The aorta and their branches were perfuse-fixed under controlled pressure 10 min after denudation. Morphometric evaluation showed dose-dependent and significant (2p < 0.01 or 0.001) inhibition of platelet spreading, adhesion and aggregation. The latter was abolished at the higher dose of SNP. Denudation and subsequent platelet adhesion caused strong vasoconstriction (2p < 0.001) which was inhibited by SNP (2p < 0.01).By exposure of subendothelium to either citrated blood or native blood in a flow chamber (2000 sec-1 shear rate) strong inhibition of spreading and adhesion-induced aggregation was again demonstrated at 6 and 12 μg/kg/min SNP. In vitro, adhesion-induced aggregation was completely abolished after the addition of SNP to rabbit (at 20 μg/ml) or human blood (2 μg/ml). 1 μg/ml PGE1 was needed to induce a similar inhibitory effect.Thus SNP is a strong inhibitor of platelet function and of injury + platelet induced vasoconstriction. These findings may explain its beneficial effect in acute myocardial infarction.

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IκB kinase 2 is not essential for platelet activation

Blood Advances ◽

10.1182/bloodadvances.2019001044 ◽

2020 ◽

Vol 4 (4) ◽

pp. 638-643

Author(s):

Manuel Salzmann ◽

Sonja Bleichert ◽

Bernhard Moser ◽

Marion Mussbacher ◽

Mildred Haase ◽

...

Keyword(s):

Platelet Activation ◽

Active Site ◽

Bleeding Time ◽

Thrombus Formation ◽

Human Platelets ◽

Iκb Kinase ◽

Specific Deletion

Abstract Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

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Talin is required for integrin-mediated platelet function in hemostasis and thrombosis

Journal of Experimental Medicine ◽

10.1084/jem.20071800 ◽

2007 ◽

Vol 204 (13) ◽

pp. 3103-3111 ◽

Cited By ~ 188

Author(s):

Brian G. Petrich ◽

Patrizia Marchese ◽

Zaverio M. Ruggeri ◽

Saskia Spiess ◽

Rachel A.M. Weichert ◽

...

Keyword(s):

Platelet Adhesion ◽

Ex Vivo ◽

Focal Adhesions ◽

Β1 Integrin ◽

Normal Morphology ◽

And Function ◽

Specific Deletion

Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin αIIbβ3-mediated platelet aggregation and β1 integrin–mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet β1 and β3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian α2β1 and αIIbβ3 integrins in vivo.

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STUDIES ON THE ANTICOAGULANT AND ANTITHROMBOTIC ACTIONS OF DERMATANS AND THEIR SULFATED DERIVATIVES

10.1055/s-0038-1643241 ◽

1987 ◽

Author(s):

D Hoppensteadt ◽

A Kumar ◽

J Fareed ◽

J Mardigian

Keyword(s):

Antithrombin Iii ◽

Femoral Vein ◽

In Vivo Studies ◽

In Vitro Assays ◽

Protease Inhibition ◽

Thrombosis Model ◽

Antithrombotic Effects ◽

Activated Prothrombin Complex Concentrates

Non-antithrombin III mediated effects such as interaction with heparin cofactor II, modulation of endothelium and polymorphonuclear leukocytes contribute to the overall antithrombotic effects of glycosaminoglycans. In order to study the role of these dermatans, we investigated their in vitro anticoagulant effects using the clot based (PT, APTT, TT, and Heptest), antiprotease (anti IIa and anti Xa) and Thromboplastin C activated fibrinopeptide A generation test. The in vivo antithrombotic actions were investigated, against activated and non activated prothrombin complex concentrates, and in combination with Russells viper venom in jugular and femoral vein stasis thrombosis models (rabbit). The dermatans studied consisted of a standard dermatan of porcine intestinal origin and four sulfated dermatans with varying degrees of sulfation. All of the dermatans studied showed weak anticoagulant effects on the routinely performed clot based assays. Marked variability was seen on the protease inhibition (anti Xa and anti IIa) assays. In the in vivo studies all dermatans studied showed varying degrees of antithrombotic actions against various thrombogenic agents in a modified stasis thrombosis model. Sulfation appeared to produce stronger anticoagulant effects as determined by in vitro assays, whereas the intravenous antithrombotic actions of native dermatan were stronger than sulfated derivatives. This data suggests that dermatans produce their antithrombotic actions via non-antithrombin III mediated pathways. Furthermore, in vitro testing methods are of limited value in the evaluation of the biologic actions of dermatans and their derivatives.

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