Identification of a substrate-like cleavage-resistant thrombin inhibitor from the saliva of the flea Xenopsylla cheopis.

SummaryRats which were infected with the gramnegative pathogen Klebsiella pneumoniae develop disseminated intravascular coagulation (DIC), multi-organ failure (MOF) and finally die in a septic shock. We investigated the therapeutic effect of antibiotic (tobramycin) treatment combined with the infusion of the highly specific thrombin inhibitor rec. hirudin. Although administration of 2 mg/kg tobramycin alone leads to a decrease of the bacterial burden, DIC could not be prevented. Infusion of rec. hirudin (0.25 mg/kg x h) for 4 h (start of treatment 1 h post infection), in addition to a bolus administration of tobramycin, led to an amelioration of DIC parameters as fibrinogen, thrombin-antithrombin complex (TAT) and platelets. Serum transaminase levels (GOT, GPT) as a marker of MOF were significantly improved by rec. hirudin, the T50 value increased from 17 h in the tobramycin group to 42 h in the tobramycin + rec. hirudin giuup, muilality rates were 90% or 60%, respectively. Combination of heparin (10011/kg x h) and tobramycin was not effective on survival.

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Increased Heparin Cofactor II Levels in Women Taking Oral Contraceptives

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647320 ◽

1990 ◽

Vol 64 (03) ◽

pp. 365-368 ◽

Cited By ~ 6

Author(s):

P Toulon ◽

J M Bardin ◽

N M Blumenfeld

Keyword(s):

Oral Contraceptives ◽

Antithrombin Iii ◽

Thrombin Inhibitor ◽

Control Groups ◽

Normal Range ◽

Heparin Cofactor Ii ◽

Men And Women ◽

Pooled Data ◽

Contraceptive Pills ◽

Increased Risk

SummaryHeparin cofactor II (HCII) is a thrombin inhibitor present in human plasma whose activity is enhanced by heparin. HCII exhibits important homologies with antithrombin III, the main heparin-enhanced thrombin inhibitor. Cases of recurrent thromboembolism have been recently reported in patients with HCII deficiency. Since the use of oral contraceptives (OC) is associated with an increased risk of thrombosis, the study of the plasma levels of HCII was undertaken in women taking contraceptive pills. Plasma HCII levels were found significantly higher in 62 women taking low-estrogen content OC (1.20 ± 0.28 U/ml) than in 62 age matched women not taking OC (0.94 ± 0.16 U/ml) or in 62 men (0.96 ± 0.19 U/ml). Significant correlations between HCII and fibrinogen levels were reported in the three groups. From the pooled data of the two control groups (men and women not taking OC), the normal range for plasma HCII levels was defined to be between 0.60 and 1.30 U/ml (mean ± 2 SD). Two cases of low HCII levels (<0.60 U/ml) were found in the control groups, but none in the group of women taking OC. It is concluded that the use of oral contraceptives is associated with a rise in HCII levels and that the screening for HCII deficiency has to be performed at distance of any OC therapy.

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The Significance of TFPI in Clotting Assays – Gomparison and Combination with other Anticoagulants

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649603 ◽

1993 ◽

Vol 70 (03) ◽

pp. 448-453 ◽

Cited By ~ 12

Author(s):

Ole Nordfang ◽

Hanne I Kristensen ◽

Sanne Valentin ◽

Per Østergaard ◽

Johnny Wadt

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Antithrombin Iii ◽

Anticoagulant Activity ◽

Assay System ◽

Thrombin Inhibitor ◽

Clotting Time ◽

Normal Plasma ◽

Tissue Factor Pathway ◽

Plasma Heparin

SummaryThe anticoagulant activities of Tissue Factor Pathway Inhibitor (TFPI), heparin and hirudin were compared in intrinsic (APTT) and extrinsic (PT) activated clotting assays. In contrast to the thrombin inhibitor hirudin, heparin was 10 fold more potent in the APTT assay than in the PT assay, indicating that inhibition of intrinsic activation is important for the anticoagulant activity of heparin as measured in an APTT assay. TFPI was most potent in the PT assay and the effect of TFPI was most pronounced in the presence of other anticoagulants (heparin and hirudin). The activities of the two natural anticoagulants antithrombin III (ATIII) and TFPI were compared in a PT assay with very dilute tissue factor. In this assay system TFPI in normal plasma affected the clotting time more than ATIII in the plasma. However, when heparin was added ATIII was the major anticoagulant, but profound Prolongation of the clotting time was only seen when TFPI was also added. In an ATIII deficient plasma heparin did not augment the effect of TFPI, showing that the increased effect of TFPI in the presence of heparin is dependent on the anticoagulant activity of ATIII/heparin. The effect of TFPI at prolonged clotting times was also illustrated by the significant effect of blocking TFPI in the plasma from warfarin-treated patients. Thus TFPI is a major anticoagulant in normal plasma and the effect of TFPI is especially seen at prolonged clotting times.

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Heparin and Low Molecular Weight Heparins Inhibit Prothrombinase Formation but not its Activity in Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648975 ◽

1994 ◽

Vol 72 (06) ◽

pp. 862-868 ◽

Cited By ~ 4

Author(s):

Frederick A Ofosu ◽

J C Lormeau ◽

Sharon Craven ◽

Lori Dewar ◽

Noorildan Anvari

Keyword(s):

Factor Xa ◽

Catalytic Efficiency ◽

Thrombin Inhibitor ◽

Lag Phase ◽

Factor V ◽

Factor X ◽

Normal Plasma ◽

Plasma Factor ◽

Prothrombin Activation ◽

Plasma Heparin

SummaryFactor V activation is a critical step preceding prothrombinase formation. This study determined the contributions of factor Xa and thrombin, which activate purified factor V with similar catalytic efficiency, to plasma factor V activation during coagulation. Prothrombin activation began without a lag phase after a suspension of coagulant phospholipids, CaCl2, and factor Xa was added to factor X-depleted plasma. Hirudin, a potent thrombin inhibitor, abrogated prothrombin activation initiated with 0.5 and 1.0 nM factor Xa, but not with 5 nM factor Xa. In contrast, hirudin did not abrogate prothrombin activation in plasmas pre-incubated with 0.5,1.0 or 5 nM α-thrombin for 10 s followed by the coagulant suspension containing 0.5 nM factor Xa. Thus, thrombin activates plasma factor V more efficiently than factor Xa. At concentrations which doubled the clotting time of contact-activated normal plasma, heparin and three low Mr heparins also abrogated prothrombin activation initiated with 0.5 nM factor Xa, but not with 5 nM factor Xa. If factor V in the factor X-depleted plasma was activated (by pre-incubation with 10 nM a-thrombin for 60 s) before adding 0.5,1.0, or 5 nM factor Xa, neither hirudin nor the heparins altered the rates of prothrombin activation. Thus, none of the five anticoagulants inactivates prothrombinase. When 5 or 10 pM relipidated r-human tissue factor and CaCl2 were added to normal plasma, heparin and the three low Mr heparins delayed the onset of prothrombin activation until the concentration of factor Xa generated exceeded 1 nM, and they subsequently inhibited prothrombin activation to the same extent. Thus, hirudin, heparin and low Mr heparins suppress prothrombin activation solely by inhibiting prothrombinase formation.

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Effects of Antithrombotic Drugs in a Rat Model of Aspirin-Insensitive Arterial Thrombosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651642 ◽

1993 ◽

Vol 69 (05) ◽

pp. 509-514 ◽

Cited By ~ 28

Author(s):

W A Schumacher ◽

T E Steinbacher ◽

C L Heran ◽

J R Megill ◽

S K Durham

Keyword(s):

Arterial Thrombosis ◽

Light And Electron Microscopy ◽

Platelet Activating Factor ◽

Thrombin Inhibitor ◽

Experimental Conditions ◽

Once Daily ◽

Thromboxane Receptor ◽

Vessel Patency ◽

Electrolytic Injury ◽

Thromboxane Receptor Antagonist

SummaryThese studies describe experimental conditions where aspirin is less effective than other antiplatelet and anticoagulant drugs in inhibiting acute arterial thrombosis. External electrolytic injury of the rat carotid artery was used to induce occlusive thrombi in 97% of vehicle-treated rats. Thrombi were revealed by light and electron microscopy to be comprised primarily of platelets enmeshed in a fibrin network. The thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl chloromethy ketone (PPACK; 6 mg/kg, i. v.) decreased thrombus weight by 90%. Aspirin alone (1, 10 and 30 mg/kg, i. v.), dipyridamole alone (5 mg/kg i. v.) and aspirin (1 and 10 mg/kg, i. v.) in combination with dipyridamole (5 mg/kg, i. v.) did not inhibit thrombosis. The platelet-activating factor (PAF) antagonist, WEB 2086, (1 mg/kg i. v.) was also ineffective. Other drugs had intermediate activity. Thrombi were decreased 56% by the thromboxane receptor antagonist, BMS 180,291, either alone (5.8 mg/kg i.v.) or in combination with aspirin (10 mg/kg, i.v.). Heparin (900 U/kg, i.v.), warfarin (0.25 mg/kg, p.o. once daily for 3 days) and ticlopidine (200 mg/ kg, p.o. once daily for 3 days) reduced thrombus weight by 63, 73 and 43% respectively. Reductions in thrombus weight were always associated with improvements in either average blood flow or vessel patency.

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Anticoagulant Activity of Hirulog™, a Direct Thrombin Inhibitor, in Humans

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651573 ◽

1993 ◽

Vol 69 (02) ◽

pp. 157-163 ◽

Cited By ~ 137

Author(s):

Irving Fox ◽

Adrian Dawson ◽

Peter Loynds ◽

Jane Eisner ◽

Kathleen Findlen ◽

...

Keyword(s):

Rational Design ◽

Therapeutic Potential ◽

Anticoagulant Activity ◽

Subcutaneous Administration ◽

Direct Thrombin Inhibitor ◽

Controlled Study ◽

Thrombin Inhibitor ◽

Thrombin Time ◽

Thrombotic Disease ◽

Dose Dependent

SummaryHirulog™ (BG8967) is a direct thrombin inhibitor built by rational design using the protein hirudin as a model (Maraganore et al. [1990]; Biochemistry 29: 7095–101). In order to evaluate the therapeutic potential for hirulog in the management of thrombotic disease, the tolerability and anticoagulant activity of the agent were examined in a study of human volunteers.In a randomized, placebo-controlled study (n = 54), the intravenous infusion of hirulog over 15 min showed a rapid, dose-dependent prolongation of activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). There was a corresponding dose-dependent increase in plasma hirulog levels. The peptide was rapidly cleared with a half-life of 36 min and a total body clearance rate for the peptide of 0.43 1 kg−1 h−1. Similar activity was observed following subcutaneous injection but with sustained pharmacodynamic and pharmacokinetic behavior. There was a significant correlation between pharmacokinetic and pharmacodynamic variables for both intravenous (r = 0.8, p <0.001) and subcutaneous administration (r = 0.7, p = 0.002).To evaluate the possible interactions of aspirin on the tolerability and anticoagulant activity of intravenous hirulog, a cross-over design was employed in eight subjects. Aspirin administration did not modify the peptide’s activity. At the administered dose of 0.6 mg kg−1 h−1 for 2 h, hirulog infusion prolonged APTT from 230 to 260% baseline. The infusion of hirulog in subjects who had received aspirin was not associated with any significant changes in the template bleeding time.The final phase of the study examined the activity and tolerability of hirulog in ten subjects during prolonged intravenous infusions for up to 24 h. The peptide (0.3 mg kg−1 h−1) exhibited sustained anticoagulant activity with no evidence for a cumulative effect. During hirulog infusion, APTT was prolonged from 210 to 250% baseline.In all phases of the study, hirulog administration was generally well-tolerated.Our observations show that hirulog is an active antithrombin agent with excellent tolerability in humans. As a direct thrombin inhibitor, hirulog provides a novel approach for the management of thrombotic disease.

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Heparin Cofactor II Deficiency in Patients Infected with the Human Immunodeficiency Virus

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649660 ◽

1993 ◽

Vol 70 (05) ◽

pp. 730-735 ◽

Cited By ~ 27

Author(s):

P Toulon ◽

M Lamine ◽

I Ledjev ◽

T Guez ◽

M E Holleman ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Absolute Number ◽

Thrombin Inhibitor ◽

Unusual Complication ◽

Healthy Individuals ◽

Heparin Cofactor Ii ◽

Immunodeficiency Virus ◽

Coagulation Inhibitor ◽

The Mean ◽

Cd4 Lymphocytes

SummaryIn human plasma, heparin cofactor II (HCII) is a thrombin inhibitor, whose deficiency has been reported to be associated with recurrent thrombosis. The finding of two cases of low plasma HCII activity in two patients infected with the human immunodeficiency virus (HIV) led us to investigate this coagulation inhibitor in the plasma of a larger population of HIV-infected patients. The mean plasma HCII activity was significantly lower in 96 HIV-infected patients than in 96 age- and sex-matched healthy individuals (0.75 ± 0.24 vs 0.99 ± 0.17 U/ml, p <0.0001). HCII antigen concentration was decreased to the same extent as the activity. The proportion of subjects with HCII deficiency was significantly higher in the HIV-infected group than in healthy individuals (38.5% vs 2.1%). In addition, HCII was significantly lower in AIDS patients than in other HIV-infected patients, classified according to the Centers for Disease Control (CDC) on the basis of an absolute number of circulating CD4+ lymphocytes below 200 x 106/1. The link between HCII and immunodeficiency is further suggested by significant correlations between HCII activity and both the absolute number of CD4+ lymphocytes and the CD4+ to CD8+ lymphocyte ratio. Nevertheless, the mean HCII level was not different in the various groups of patients classified according to clinical criteria, except in CDC IVD patients in whom HCII levels were significantly lower. In addition, no correlation could be demonstrated between HCII and protein S activities, another coagulation inhibitor whose plasma level was also found to be decreased in HIV-infected patients. A similar prevalence of HCII deficiency was also found in a small series of 7 HIV-infected patients who developed thrombotic episodes, an unusual complication of the infection. This suggests that, in HIV-infected patients, HCII deficiency is not in itself the causative factor for the development of thrombosis.

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Inhibition of Thrombin Generation in Plasma by Inhibitors of Factor Xa

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657717 ◽

1997 ◽

Vol 78 (04) ◽

pp. 1215-1220 ◽

Cited By ~ 25

Author(s):

D Prasa ◽

L Svendsen ◽

J Stürzebecher

Keyword(s):

Thrombin Generation ◽

Factor Xa ◽

Thrombin Inhibitors ◽

Thrombin Inhibitor ◽

Thrombin Generation Assay ◽

Ic50 Values ◽

Fxa Inhibitor ◽

20 Nm ◽

Tick Anticoagulant Peptide ◽

Inhibition Of Thrombin

SummaryA series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations.The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.

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Combined Thrombin and Platelet Inhibition Treatment for HIT Patients

Hämostaseologie ◽

10.1055/s-0038-1660400 ◽

1999 ◽

Vol 19 (03) ◽

pp. 128-133 ◽

Cited By ~ 1

Author(s):

B.E. Lewis ◽

W. P. Jeske ◽

F. Leya ◽

Diane Wallis ◽

M. Bakhos ◽

...

Keyword(s):

Standard Dose ◽

Combined Therapy ◽

Platelet Inhibition ◽

Thrombin Inhibitors ◽

Thrombin Inhibitor ◽

Heparin Induced Thrombocytopenia ◽

Acute Thrombosis ◽

Anti Platelet Drug ◽

Receptor Inhibitor

SummaryDespite the use of potent anticoagulants such as r-hirudin and argatroban, the morbidity and mortality in heparin-induced thrombocytopenia (HIT) patients remains unacceptable. Data from our in vitro investigations show that thrombin inhibitors do not block platelet activation induced by heparin antibodies and heparin but that GPIIb/IIIa receptor inhibitors do block this process. We have treated four HIT positive patients with a combined therapy of thrombin inhibitor and GPIIb/IIIa receptor inhibitor when treatment with thrombin inhibitor alone failed to alleviate acute thrombosis. Combination therapies included r-hirudin (Refludan®) with tirofiban (Aggrastat®) and argatroban (Novastan®) with abciximab (ReoPro®). A reduced dose of the thrombin inhibitor was used with the standard dose of the anti-platelet drug. In all cases, there was no overt bleeding which required intervention, and all patients exhibited clinical improvement or full recovery. These case studies suggest that treatment of active thrombosis in HIT patients with adjunct GPIIb/IIIa receptor inhibitor therapy may be more effective than thrombin inhibitor treatment alone.

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Factor X Activation by washed human platelets

10.1055/s-0038-1665669 ◽

1979 ◽

Author(s):

E van Wijk ◽

L Kahlé ◽

J ten Cate

Keyword(s):

Human Factors ◽

Trypsin Inhibitor ◽

Calcium Ions ◽

Factor Xa ◽

Human Platelets ◽

Soybean Trypsin Inhibitor ◽

Thrombin Inhibitor ◽

Chromogenic Substrate ◽

Factor X ◽

Factor X Activation

In a system of washed human platelets, Ca2+and purified human factors X anc II, a sufficient amount of thrombin is generated in about 10 minutes to aggregate the platelets. This thrombin is formed through the activation of FX by the platelets. In a system with either FX or FII present, no aggregation occurs. In addition no aggregation is observed when hirudin, a specific thrombin inhibitor, or when soybean trypsin inhibitor, which inhibits factor Xa, are added to the mixture. The formation of factor Xa can be monitored indirectly through the generation of thrombin, in the presence of an excess of prothrombin, using a thrombin sensitive chromogenic substrate. When washed platelets are incubated with FX alone for 10 minutes, no aggregation occurs and after the addition of prothrombin aggregation starts within 6 minutes. These findings confirm that washed platelets possess a factor X activating property. The generation of FXa proceeds in the absence of added Ca2+, whereas in the presence of Ca2+factor Xa activity reaches a maximum in 3 minutes, whereafter the activity progressively decreases. This may be due to the binding of Xa to the platelets in the presence of calcium ions.

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