An enzyme-linked immunosorbent assay for heparin cofactor II (HCII). Application to the measurement of HCII in clinical materials.

Heparin cofactor II (HCII) is a thrombin inhibitor in human plasma, the activity of which is enhanced by heparin and dermatan sulfate. To measure the plasma antigen concentration of HCII, an enzyme-linked immunosorbent assay (ELISA) has been developed, and this is based on the use of polyclonal anti-HCII IgG, both as the antigen-capturing antibody and as the labelled antibody. The intra- and inter-assay reproducibilities have been calculated to be less than 6%. HCII antigen concentrations evaluated with this ELISA technique in clinical plasma samples correlated well with those determined by electroimmunodiffusion and with HCII activities evaluated using a functional assay (r = 0.909 and r = 0.930, P less than 0.0001). Since the correlation between HCII deficiency and thrombosis is still a controversial issue, the ELISA technique described in this report could be a useful tool for the large-scale studies needed to determine the prevalence of HCII deficiency in healthy individuals and in patients with a history of thrombosis.     

An anticoagulant dermatan sulfate proteoglycan circulates in the pregnant woman and her fetus.

Investigation of the in vitro ability of plasma from pregnant women to inhibit exogenous thrombin (25 nM) demonstrated that heparin cofactor II inhibited more thrombin (3.0 +/- 0.7 nM, mean +/- SD) than plasma from women 3-5 d postpartum (1.9 +/- 0.5 nM) or plasma from nonpregnant adults (1.5 +/- 0.4 nM). Levels of heparin cofactor II were only slightly increased over normal in both pregnant and postpartum women and did not account for the observed increase in thrombin bound to heparin cofactor II. Assay of pregnancy plasma for dermatan sulfate anticoagulant activity demonstrated the presence of activity equivalent to 0.23 +/- 0.02 micrograms/ml of porcine mucosal dermatan sulfate. This activity could not be demonstrated in normal adult plasma or plasma from women on the contraceptive pill. The mass of dermatan sulfate in pregnancy and umbilical cord plasmas was increased over adult control plasma by 0.20 micrograms/ml (53%) and 0.29 micrograms/ml (76%), respectively. The glycosaminoglycan-containing fraction of plasma was isolated and an assay for anticoagulant dermatan sulfate confirmed its presence in both pregnancy and cord plasmas but minimal activity in adult plasma. Gel chromatography of isolated fractions from both pregnancy and cord plasmas revealed a polydisperse, active species with apparent Mr 150,000 D. Reductive elimination decreased the apparent Mr of the active species on gel chromatography to 31,000 D for cord and 21,000 D for pregnancy products. This confirmed the presence of an anticoagulant active dermatan sulfate proteoglycan circulating in the plasmas of pregnant women at term and fetuses at delivery.     

Modulation of heparin cofactor II activity by histidine-rich glycoprotein and platelet factor 4.

Heparin cofactor II is a plasma protein that inhibits thrombin rapidly in the presence of either heparin or dermatan sulfate. We have determined the effects of two glycosaminoglycan-binding proteins, i.e., histidine-rich glycoprotein and platelet factor 4, on these reactions. Inhibition of thrombin by heparin cofactor II and heparin was completely prevented by purified histidine-rich glycoprotein at the ratio of 13 micrograms histidine-rich glycoprotein/microgram heparin. In contrast, histidine-rich glycoprotein had no effect on inhibition of thrombin by heparin cofactor II and dermatan sulfate at ratios of less than or equal to 128 micrograms histidine-rich glycoprotein/microgram dermatan sulfate. Removal of 85-90% of the histidine-rich glycoprotein from plasma resulted in a fourfold reduction in the amount of heparin required to prolong the thrombin clotting time from 14 s to greater than 180 s but had no effect on the amount of dermatan sulfate required for similar anti-coagulant activity. In contrast to histidine-rich glycoprotein, purified platelet factor 4 prevented inhibition of thrombin by heparin cofactor II in the presence of either heparin or dermatan sulfate at the ratio of 2 micrograms platelet factor 4/micrograms glycosaminoglycan. Furthermore, the supernatant medium from platelets treated with arachidonic acid to cause secretion of platelet factor 4 prevented inhibition of thrombin by heparin cofactor II in the presence of heparin or dermatan sulfate. We conclude that histidine-rich glycoprotein and platelet factor 4 can regulate the antithrombin activity of heparin cofactor II.     

Fibrin moderates the catalytic action of heparin but not that of dermatan sulfate on thrombin inhibition in human plasma

Three recent studies have reported that fibrin in solution significantly inhibits the ability of heparin to catalyze the inhibition of thrombin by antithrombin III. In addition, heparin inhibits the release of fibrinopeptide A by clot-bound thrombin less effectively than it inhibits the release of fibrinopeptide A by thrombin in solution. We have also reported that dermatan sulfate, which catalyzes thrombin inhibition by heparin cofactor II, inhibits thrombus growth in rabbits more effectively than heparin. Because the results of these studies suggest that fibrin inhibits the reactivity of thrombin with antithrombin III-heparin but not with heparin cofactor II-dermatan sulfate, we compared the relative catalytic effects of heparin and dermatan sulfate on thrombin inhibition in plasma, both in the presence and absence of fibrin. We quantitated the rates of thrombin inhibition by antithrombin III and heparin cofactor II by specific enzyme-linked immunosorbent assays. When it was generated, fibrin was kept in solution by adding 2 mmol/L Gly-Pro-Arg-Pro to plasma. Fibrinogen-fibrin reduced the reactivity of thrombin with plasma antithrombin III, both in the presence of and in the absence of heparin. In contrast, the catalytic action of dermatan sulfate on thrombin inhibition by plasma heparin cofactor II was unimpaired by fibrinogen-fibrin. Based on the ability of dermatan sulfate to inhibit thrombus growth in rabbits, failure of fibrinogen-fibrin to moderate the catalytic action of dermatan sulfate may account for its greater antithrombotic effectiveness relative to that of heparin.     

Ascidian dermatan sulfates attenuate metastasis,inflammation and thrombosis by inhibition of P‐selectin

See also Zacharski LR. Controlling cancer growth from within the blood coagulation mechanism. This issue, pp 1804–6. Summary. Background: Cancer‐associated thrombosis and enduring inflammation are strongly associated with cancer progression and metastasis. Heparin is the mostly clinically used anticoagulant/antithrombotic drug, and has recently been shown to exhibit antimetastatic and anti‐inflammatory activities that are linked to inhibition of P‐selectin and/or L‐selectin. P‐selectin‐mediated platelet–tumor cell and tumor cell–endothelium interactions facilitate the initial steps of metastasis. Objectives and Methods: The aim of the present study was to determine the capacity of dermatan sulfates to inhibit P‐selectin and to test their potential to affect thrombosis, inflammation and metastasis in respective experimental mouse models. Results: Two dermatan sulfates isolated from the ascidians Styela plicata and Phallusia nigra, composed of the same disaccharide core structure (IdoA2‐GalNAc)_n, but sulfated at carbon 4 or 6 of the GalNAc, respectively, have opposed heparin cofactor II (HCII) activities and are potent inhibitors of P‐selectin. The ascidian dermatan sulfates effectively attenuated metastasis of both MC‐38 colon carcinoma and B16‐BL6 melanoma cells and the infiltration of inflammatory cells in a thioglycollate peritonitis mouse model. Moreover, both glycosaminoglycans reduced thrombus size in an FeCl₃‐induced arterial thrombosis model, irrespective of their HCII activities. The analysis of arterial thrombi demonstrated markedly reduced platelet deposition after dermatan sulfate treatment, suggesting that the glycosaminoglycan inhibited P‐selectin and thereby the binding of activated platelets during thrombus formation. Conclusions: Collectively, these findings provide evidence that specific inhibition of P‐selectin represents a potential therapeutic target in thrombosis, inflammation and metastasis, and that ascidian dermatan sulfates may serve as antiselectin agents.     

Normal levels of anticoagulant heparan sulfate are not essential for normal hemostasis

Endothelial cell production of anticoagulant heparan sulfate (HS(act)) is controlled by the Hs3st1 gene, which encodes the rate-limiting enzyme heparan sulfate 3-O-sulfotransferase-1 (3-OST-1). In vitro, HS(act) dramatically enhances the neutralization of coagulation proteases by antithrombin. The in vivo role of HS(act) was evaluated by generating Hs3st1(-/-) knockout mice. Hs3st1(-/-) animals were devoid of 3-OST-1 enzyme activity in plasma and tissue extracts. Nulls showed dramatic reductions in tissue levels of HS(act) but maintained wild-type levels of tissue fibrin accumulation under both normoxic and hypoxic conditions. Given that vascular HS(act) predominantly occurs in the subendothelial matrix, mice were subjected to a carotid artery injury assay in which ferric chloride administration induces de-endothelialization and occlusive thrombosis. Hs3st1(-/-) and Hs3st1(+/+) mice yielded indistinguishable occlusion times and comparable levels of thrombin.antithrombin complexes. Thus, Hs3st1(-/-) mice did not show an obvious procoagulant phenotype. Instead, Hs3st1(-/-) mice exhibited genetic background-specific lethality and intrauterine growth retardation, without evidence of a gross coagulopathy. Our results demonstrate that the 3-OST-1 enzyme produces the majority of tissue HS(act). Surprisingly, this bulk of HS(act) is not essential for normal hemostasis in mice. Instead, 3-OST-1-deficient mice exhibited unanticipated phenotypes suggesting that HS(act) or additional 3-OST-1-derived structures may serve alternate biologic roles.     

Pharmacologic properties of a low molecular weight dermatan sulfate: comparison with unfractionated dermatan sulfate

The anticoagulant, pharmacodynamic, and antithrombotic properties of a low molecular weight dermatan sulfate (molecular weight range 1600 to 8000, peak 4000) were compared with those of unfractionated dermatan sulfate (molecular weight range 12,000 to 45,000, peak 25,000). Anticoagulant activities were evaluated as the ability of the compounds to catalyze the inhibition of thrombin in the presence of heparin cofactor II in a purified system and to prolong the activated partial thromboplastin time or the thrombin clotting time of human and rabbit plasmas. On the basis of weight, low molecular weight dermatan sulfate was two times less potent than unfractionated dermatan sulfate. After bolus intravenous injection into rabbits, the volume of distribution of low molecular weight dermatan sulfate was 10 times larger than that of unfractionated compound, and the half-life of disappearance was two to four times longer despite a 1.4 to 2.3 times higher total clearance. The bioavailability of low molecular weight dermatan sulfate from its subcutaneous depot was 100%; it was absorbed faster from that depot than unfractionated dermatan sulfate. The antithrombotic activities of unfractionated and of low molecular weight dermatan sulfate were also examined with a Wessler-type model with tissue factor as the thrombogenic stimulus. When evaluated 3 minutes after a bolus intravenous injection, unfractionated dermatan sulfate was twice as active as low molecular weight dermatan sulfate on the basis of weight. With subcutaneous injection, 10 mg/kg of low molecular weight dermatan sulfate generated an activity in plasma equivalent to 5.6 micrograms/ml 1 hour later. This concentration was associated with a significant antithrombotic effect that lasted for less than 6 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

血栓疾病患者血浆肝素辅因子Ⅱ活性和抗原水平检测及意义

目的　了解肝素辅因子Ⅱ (HCⅡ )活性及抗原水平在动、静脉血栓性疾病中的变化及其与动、静脉血栓性疾病之间的关系。方法　用发色底物法测定 5 0名正常人 ,75例脑梗死、5 0例心肌梗死及 36例深静脉血栓患者血浆HCⅡ活性 ;发色底物法检测深静脉血栓患者中HCⅡ缺乏者血浆抗凝血酶 (AT)活性 ;Westernblot检测部分样品 (每组 36例 )血浆HCⅡ抗原含量。结果　血浆HCⅡ活性与其抗原呈平行变化 ;正常对照组血浆HCⅡ活性及抗原水平分别为 (96 .80± 2 0 .11) % ,0 .93± 0 .19与脑梗死组 [(99.97± 2 1.14 ) % ,0 .96± 0 .2 4 ]、心肌梗死组 [(98.18± 2 9.35 ) % ,0 .95± 0 .2 0 ]及深静脉血栓形成组 [(89.5 7± 17.12 ) % ,0 .87± 0 .18]比较 ,差异无显著性 ,但深静脉血栓形成组HCⅡ活性和抗原均呈降低趋势 ;HCⅡ明显减低者在正常人及患者之间的分布频度无显著差异 ;深静脉血栓中HCⅡ缺乏者血浆抗凝血酶活性及纤维蛋白原浓度正常。结论　血浆HCⅡ变化可能不是中国湖南汉族人心、脑血栓病的危险因子 ;是否与静脉血栓形成相关有待进一步证实。     

Strain-dependent embryonic lethality and exaggerated vascular remodeling in heparin cofactor II-deficient mice

Heparin cofactor II (HCII) specifically inhibits thrombin action at sites of injured arterial wall, and patients with HCII deficiency exhibit advanced atherosclerosis. However, the in vivo effects and the molecular mechanism underlying the action of HCII during vascular remodeling remain elusive. To clarify the role of HCII in vascular remodeling, we generated HCII-deficient mice by gene targeting. In contrast to a previous report, HCII(-/-) mice were embryonically lethal. In HCII(+/-) mice, prominent intimal hyperplasia with increased cellular proliferation was observed after tube cuff and wire vascular injury. The number of protease-activated receptor-1-positive (PAR-1-positive) cells was increased in the thickened vascular wall of HCII(+/-) mice, suggesting enhanced thrombin action in this region. Cuff injury also increased the expression levels of inflammatory cytokines and chemokines in the vascular wall of HCII(+/-) mice. The intimal hyperplasia in HCII(+/-) mice with vascular injury was abrogated by human HCII supplementation. Furthermore, HCII deficiency caused acceleration of aortic plaque formation with increased PAR-1 expression and oxidative stress in apoE-KO mice. These results demonstrate that HCII protects against thrombin-induced remodeling of an injured vascular wall by inhibiting thrombin action and suggest that HCII is potentially therapeutic against atherosclerosis without causing coagulatory disturbance.     

刺参酸性粘多糖介导肝素辅因子Ⅱ对凝血酶活性的抑制

目的：进一步澄清刺参酸性粘多糖（Ｓｊａｍｐ）的抗凝血酶机制。方法：用国产Ｓｊａｍｐ作为激动剂，在正常混合血浆体系、纯化的肝素辅因子Ⅱ（ＨＣⅡ）体系以及纯化的抗凝血酶Ⅲ（ＡＴ－Ⅲ）体系研究Ｓｊａｍｐ的抗凝血酶作用机制。结果：Ｓｊａｍｐ对凝血酶的抑制主要呈ＨＣⅡ依赖性，当存在ＨＣⅡ时，Ｓｊａｍｐ抗凝血酶作用的二级速率常数Ｋ２＝１．５６×１０７ｍ－１·ｍｉｎ－１，其抑制速率常数是ＡＴ－Ⅲ的４．６倍。结论：在抗凝血酶作用方面，Ｓｊａｍｐ的效率（Ｋ２值）及机制（ＨＣⅡ依赖性）与硫酸皮肤素类似。     

Mechanisms of glycosaminoglycan activation of the serpins in hemostasis

Summary.  Serpins are the predominant protease inhibitors in the higher organisms and are responsible, in humans, for the control of many highly regulated processes including blood coagulation and fibrinolysis. The serpin inhibitory mechanism has recently been revealed by the solution of a crystallographic structure of the final serpin–protease complex. The serpin mechanism, in contrast to the classical lock-and-key mechanism, involves dramatic conformational change in both the inhibitor and the inhibited protein. The final result is a stable covalent complex in which the properties of each component are altered so as to allow clearance from the circulation. Several serpins are involved in hemostasis: antithrombin (AT) inhibits many coagulation proteases, most importantly factor Xa and thrombin; heparin cofactor II (HCII) inhibits thrombin; protein C inhibitor (PCI) inhibits activated protein C and thrombin bound to thrombomodulin; plasminogen activator inhibitor 1 inhibits tissue plasminogen activator; and α₂-antiplasmin inhibits plasmin. Nearly all of these reactions are accelerated through interactions with glycosaminoglycans (GAGs) such as heparin or heparan sulfate. Recent structures of AT, HCII and PCI have revealed how in each case the serpin mechanism has been fine-tuned by evolution to bring about high levels of regulatory control, and how seemingly disparate mechanisms of GAG binding and activation can share critical elements. By considering the serpins involved in hemostasis together it is possible to develop a deeper understanding of their complex individual roles.     

妊娠高血压综合征分娩前,后血浆肝素辅助因子Ⅱ的动态变化？…

目的 揭示肝素辅助因子Ⅱ（ＨＣⅡ）体内抗凝的生理意义,探讨妊娠高血压综合征（妊高征）并发血栓形成时的部分发病机制。方法 （１）采用发色底物法测定１８名正常人,１８例正常妊娠及２９例妊高征分娩前,后血浆ＨＣⅡ活性。（２）用Ｗｅｓｔｅｒｎ印迹法检测２９例妊高征分娩前,后血浆ＨＣⅡ抗原;（３）免疫荧光定位检测ＨＣⅡ在胎盘组织上的分布。结果 妊高征产前血浆ＨＣⅡ活性及抗原含量显降低,其降低程度与妊高征分     

A rationale for targeting antithrombotic therapy at the vessel wall: improved antithrombotic effect and decreased risk of bleeding

Intimal hyperplasia after percutaneous transluminal coronary angioplasty (PTCA) or vascular surgical procedures remains a significant problem despite current antithrombotic therapy. The use of the current antithrombotic drugs, namely heparin + chronic aspirin (ASA) +/- oral anticoagulants, is based upon the assumptions that: i) heparin blocks thrombin generation and/or accelerates thrombin inhibition by antithrombin III (ATIII); ii) aspirin acetylates platelet cyclooxygenase, thereby preventing thromboxane A2 (TxA2) synthesis; and iii) oral anticoagulants reduce the availability of vitamin K-dependent procoagulants, thereby reducing the risk of thrombus formation. Albeit beneficial, this approach has a number of shortcomings and limitations: i) when thrombin binds to an injured vessel wall, it becomes resistant to inhibition by heparin/ATIII; thus, surface-bound thrombin remains active, stimulating further thrombus formation, smooth muscle cell proliferation and subsequent hyperplasia; ii) while TxA2 inhibition reduces platelet reactivity, platelets are able to respond to multiple stimuli generated at the time of, or after, vessel wall injury; and iii) heparin, aspirin and the oral anticoagulants all render the patient hemostatically defective and at risk of bleeding. Recent studies suggest that alternate therapeutic approaches can inhibit thrombogenesis more effectively at the time of injury, thereby not only inhibiting hyperplasia more effectively than the currently used drugs, but also reducing (or eliminating) the need for long-term therapy. For example, we suggest that the heparin cofactor II (HCII) catalysts, dermatan sulfate and Intimatan, inhibit surface-bound thrombin more effectively than heparin/ATIII, thereby inhibiting intimal hyperplasia effectively. Their effects are achieved when the drug is given only at the time of injury; i.e. with no further antithrombotic therapy. Other studies indicate that injured vessel wall thrombogenicity can be reduced by pretreatment with Persantine (dipyridamole) or with certain fatty acid supplements which either increase vessel wall cAMP and/or 13HODE synthesis. These increases are associated with decreased vessel wall thrombogenicity, which, in turn, is associated with decreased intimal hyperplasia. Such results suggest that vessel wall repair is achieved more effectively by targeting antithrombotic drugs directly at the vessel wall thrombogenicity per se rather than indirectly by altering the circulating blood cells and systemic coagulant system.     

Development of a calibrated automated thrombography based thrombin generation test in mouse plasma

BACKGROUND: Mouse models have become increasingly important in thrombosis research. However, only a limited number of assays are available for assessment of the coagulation system in mouse plasma. OBJECTIVES: To quantify tissue factor-initiated thrombin generation in murine platelet-rich and platelet-free plasma and to develop a test for measurement of resistance to activated protein C (APC) in mouse plasma. METHODS: Thrombin generation was monitored with calibrated automated thrombography (CAT) using a low-affinity fluorogenic substrate for thrombin. RESULTS: To overcome the higher activity of coagulation inhibitors in mouse plasma as compared with human plasma, the reaction temperature was lowered to 33 degrees C and the assay was carried out at a 2-fold higher final plasma dilution (1:3) than commonly used for CAT in human plasma. This increased the endogenous thrombin potential (ETP) 4- to 5-fold and enabled reliable measurement of thrombin generation in both platelet-free and platelet-rich mouse plasma. For the APC resistance measurement, the reaction conditions were further optimized with respect to tissue factor, phospholipid, APC and CaCl(2) concentrations. The test was validated using plasma of mice with different genetic background with respect to the factor V Leiden mutation (FV Leiden). Mice homozygous for FV Leiden had higher APC sensitivity ratios (mean 5.46; 95% CI 4.88-6.03) than heterozygous FV Leiden mice (mean 4.21; 95% CI 3.53-4.89) and than wild-type mice (mean 2.71; 95%CI 2.15-3.27). CONCLUSIONS: We have established reaction conditions for measurement of thrombin generation and APC resistance in mouse plasma. This assay enables evaluation of the coagulation system and the function of the protein C system in mouse models.     

Pregnancy-associated changes in the hemostatic system in wild-type and factor V Leiden mice

Summary.  Background:  Pregnancy, oral contraceptive (OC) use and hormone replacement therapy (HRT) are established risk factors for venous thrombosis. Acquired resistance to activated protein C (APC) has been proposed to contribute to the increased thrombosis risk. Mouse models are often used for preclinical testing of newly developed hormone preparations. However, it is not known whether hormone-induced APC resistance is also observed in laboratory animals. Objectives:  To investigate whether hormonal changes modulate APC resistance in mice, we used pregnant mice as a model of hormone-induced APC resistance. The effect of pregnancy on APC resistance was studied in wild-type and factor (F)V Leiden mice. Methods:  APC resistance was determined in mouse plasma using a thrombin generation-based APC resistance test. APC resistance determinants, i.e. prothrombin, FV, FX, antithrombin and protein S levels, and of tissue factor pathway inhibitor (TFPI) activity were evaluated in plasma from non-pregnant and pregnant mice. Results:  In contrast to humans, pregnancy induced a decrease in APC resistance in wild-type and in FV Leiden mice. Pregnant mice had higher levels of prothrombin, FV, FX, protein S and TFPI activity as compared with non-pregnant mice. Conclusions:  Pregnancy causes a decrease in APC resistance in mice, which can be explained by the elevation of protein S levels and increased TFPI activity in plasma. Our findings show species specificity in the effects of pregnancy on the major determinants of the protein C system and suggest that protein S and TFPI play an important role in the development of pregnancy-induced APC resistance in humans.     

A comparison of the β-D-xyloside, odiparcil, to warfarin in a rat model of venous thrombosis

BACKGROUND: A significant need exists for new chronic oral anticoagulation therapies to replace warfarin. Previous studies have shown that beta-D-xylosides, which prime glycosaminoglycan (GAG) synthesis, have antithrombin and antithrombotic activity. In the following report, a new orally active beta-D-xyloside (odiparcil) has been characterized in a rat model of venous thrombosis and its efficacy and bleeding liability compared to warfarin. Additionally, studies were conducted to investigate odiparcil's ex vivo antithrombin and antiplatelet activity, and also to explore the potential utility of protamine sulfate as a neutralizing agent. METHODS AND RESULTS: In vivo thrombosis studies were conducted in a rat inferior vena cava model, and bleeding studies in a rat tail transection model. Following oral dosing, warfarin and odiparcil produced dose-related suppression of thrombus formation. A therapeutically relevant dose of warfarin in this model (international normalized ratio; INR 3.0) achieved approximately 65% inhibition of thrombus formation. Warfarin caused dose-related significant increases in bleeding indices. Odiparcil antithrombotic activity was limited by its mechanism to a maximum suppression of thrombus formation of 65-70%, and did not prolong bleeding indices. Additionally, odiparcil-induced heparin cofactor II (HCII)-dependent antithrombin activity was shown to be a function of dermatan sulfate-like GAG production. Other than thrombin-related effects, no odiparcil effects on platelet function were observed. In antidote studies, it was demonstrated that odiparcil-induced antithrombotic activity could be partially neutralized by protamine sulfate. CONCLUSIONS: These experiments suggest that an antithrombotic approach based upon xyloside induction of circulating GAGs may have the potential to approximate the efficacy of warfarin and yet with a reduced risk to hemostasis.     

Neutralization of the anticoagulant effects of glycosaminoglycans by serum amyloid P component: comparison with other plasma and platelet proteins.

Serum amyloid P protein (SAP) is a heparin-binding protein that is found in blood and connective tissues including some types of vascular basement membrane. In this article we present evidence that SAP is capable of blocking the anticoagulant effects of glycosaminoglycans. SAP neutralized the catalytic effect of heparin on the thrombin-antithrombin III reaction more effectively than vitronectin, histidine-rich glycoprotein, fibronectin, and high-molecular-weight kininogen and almost as effectively as platelet factor 4. SAP also blocked the effects of heparin and dermatan sulfate on the inhibition of thrombin by heparin cofactor II. We found evidence for the formation of a high-affinity 1:1 complex between SAP and heparin and for inhibition of binding of both thrombin and antithrombin III to heparin-Sepharose by SAP. We conclude that SAP may account for much of the heparin-neutralizing capacity of plasma under some conditions and that basement-membrane-bound SAP may modulate extravascular coagulation by blocking the anticoagulant effects of basement membrane glycosaminoglycans.     

Pharmacological interruption of acute thrombus formation with minimal hemorrhagic complications by a small molecule tissue factor/factor VIIa inhibitor: comparison to factor Xa and thrombin inhibition in a nonhuman primate thrombosis model

This study was designed to evaluate the antithrombotic efficacy and bleeding propensity of a selective, small-molecule inhibitor of tissue factor/factor VIIa (TF/VIIa) in comparison to small-molecule, selective inhibitors of factor Xa and thrombin in a nonhuman primate model of thrombosis. Acute, spontaneous thrombus formation was induced by electrolytic injury to the intimal surface of a femoral blood vessel, which results in thrombus propagation at the injured site. The TF/FVIIa inhibitor 3-amino-5-[1-[2-([4-[amino(imino)methyl]benzyl]amino)-2-oxoethyl]-3-chloro-5-(isopropylamino)-6-oxo-1,6-dihydropyrazin-2-yl]benzoic acid dihydrochloride (PHA-927F) was fully effective in prevention of thrombosis-induced vessel occlusion at a dose of 400 microg/kg/min, i.v., in the arterial vasculature (femoral artery). Neither the effective dose nor multiples up to 4.4-fold the effective arterial plasma concentration elicited any significant effect on bleeding time or blood loss from either the bleeding time site or the surgical (femoral isolation) site. Small-molecule inhibitors of factor Xa or thrombin were effective arterial antithrombotic agents; however, in contrast to the TF/FVIIa inhibitor, they both elicited substantial increases in bleeding propensity at the effective dose and at multiples of the effective plasma concentration. These data indicate that TF/VIIa inhibition effectively prevented arterial thrombosis with less impact on bleeding parameters than equivalent doses of factor Xa and thrombin inhibitors.     

Identification of a congenital dysthrombin, thrombin Quick.

A dysprothrombin designated prothrombin Quick, is isolated from the plasma of an individual with < 2% of normal functional prothrombin activity and 34% of the normal prothrombin level by immunologic assay. With Factor Xa or taipan snake venom as activators, a fragmentation pattern identical to that of normal prothrombin is observed on gel electrophoresis in dodecylsulfate. This evidence combined with the observed barium citrate adsorption of prothrombin Quick and the low activity suggests that the defect in prothrombin Quick is in the thrombin portion of the molecule. Thrombin Quick is isolated and comigrates with thrombin on dodecyl sulfate gel electrophoresis, either reduced or nonreduced. The activity of thrombin Quick on several biological substrates of thrombin is investigated. Relative to normal thrombin, thrombin Quick is 1/200 as active on fibrinogen and 1/20-1/50 as effective in activating Factors V and VIII and aggregating platelets. A complex with antithrombin III is detected by dodecyl sulfate gel electrophoresis. Further investigation with the active site titrant, dansylanginine-N-(3-ethyl-1,5-pentanediyl)amide showed that the thrombin Quick preparation has the same affinity for the titrant as thrombin, but apparently only 40% active sites per mole protein are titrable.     

Intimatan prevents arterial and venous thrombosis in a canine model of deep vessel wall injury

Resistance of fibrin-bound thrombin to inactivation by the heparin/antithrombin III complex is considered a limitation in the use of heparin as an antithrombotic agent. Intimatan (dermatan 4,6-di-O-sulfate) is a heparin cofactor II agonist that inhibits both free and bound forms of thrombin. The present study examines the hypothesis that Intimatan prevents thrombotic occlusion in response to vascular wall injury in a canine model of carotid artery/jugular vein thrombosis. The left carotid artery and right jugular vein served as vehicle-treated control vessels, whereas the right carotid artery and left jugular vein were subjected to electrolytic injury after administration of Intimatan (9 mg/kg bolus + 300 microg/kg/min infusion, i.v.) or dalteparin (Fragmin) (400 IU/kg, s.c.). Intimatan significantly increased time to carotid artery (226.0 +/- 14.0 min) and jugular vein (240.0 +/- 0.0 min) thrombosis, compared with control vessels (carotid artery, 87.1 +/- 7.9 min; jugular vein, 60.6 +/- 7.4 min). Vessel patency was maintained in eight of eight jugular veins and seven of eight carotid arteries during treatment with Intimatan. Dalteparin significantly increased time to carotid artery thrombosis (122.1 +/- 17.5 min) compared with control (64.3 +/- 8.2 min), but did not change the time to thrombosis in the jugular vein. Only one carotid artery remained patent at the end of the dalteparin protocol. The two drugs produced minimal increases in bleeding times, and Intimatan increased the activated partial thromboplastin time above that observed with dalteparin. The results demonstrate that Intimatan is effective in preventing occlusive arterial and venous thrombosis in an experimental model of deep vascular wall injury.