Heparin cofactor II: discovery, properties, and role in controlling vascular homeostasis

Heparin cofactor II (HCII) is a serine protease inhibitor (serpin) found in high concentrations in human plasma. Despite its discovery >30 years ago, its physiological function is still poorly understood. It is known to inhibit thrombin, the predominant coagulation protease, and HCII-thrombin complexes have been found in plasma, yet it is thought to contribute little to normal hemostasis. However, thrombin has several other physiological functions, and therefore many biological roles for HCII need consideration. The unique structure and mechanism of action of HCII have helped guide our understanding of HCII. In particular, HCII binds many glycosaminoglycans (GAGs) such as heparin and heparin sulfate as well as several different polyanions to enhance its inhibition of thrombin. Distinctly, HCII is able to use the GAG dermatan sulfate for accelerated thrombin inhibition. Dermatan sulfate is found in high concentrations in the walls of blood vessels as well as in placental tissue. This knowledge has led to research indicating that HCII may play a protective role in atherosclerosis and placental thrombosis. Additionally, pharmaceuticals are being developed that use the dermatan sulfate activation of HCII for anticoagulation. Although much research is still needed to fully understand HCII, this humble protein may have significant impact in our medical future. This article reviews the laboratory history, protein characteristics, structure-activity relationships, protease inhibition, physiological function, and medical relevance of HCII in hopes of regenerating interest in this sometimes forgotten serpin.     

Antithrombotic activity of dermatan sulfate in heparin cofactor II-deficient mice

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. We previously reported that the time to thrombotic occlusion of the carotid artery after photochemical injury was shorter in HCII-deficient mice than in wild-type control animals. In this paper, we describe the antithrombotic activity of dermatan sulfate in wild-type and HCII-deficient mice. Intravenous administration of porcine skin dermatan sulfate induced a dose-dependent prolongation of the carotid artery occlusion time in HCII(+/+) mice that was not observed in HCII(-/-) animals. Pharmacokinetic studies suggested that porcine skin dermatan sulfate expresses antithrombotic activity after being transferred from the plasma to sites in the vessel wall. Using invertebrate dermatan sulfate preparations, we showed that N-acetylgalactosamine-4-O-sulfate residues are required for the HCII-dependent antithrombotic effect. Furthermore, the invertebrate dermatan sulfates, which have higher charge densities than mammalian dermatan sulfate, slightly prolonged the thrombotic occlusion time of HCII(-/-) mice. These results indicate that HCII mediates the antithrombotic effect of porcine skin dermatan sulfate after injury to the carotid arterial endothelium in mice, whereas more highly charged dermatan sulfates possess weak antithrombotic activity independent of HCII.     

Heparin cofactor II: an acute phase reactant in patients with deep vein thrombosis.

In human plasma, heparin cofactor II (HCII) is a thrombin inhibitor which displays similarities with antithrombin III (ATIII). As previously reported for hereditary ATIII deficiency, cases of recurrent thrombosis were reported in patients with hereditary HCII deficiency. Here, plasma HCII activity was studied in 372 patients with a history of thrombosis, classified according to their anticoagulant therapy. The mean plasma HCII level was significantly higher in patients with acute deep vein thrombosis (DVT) under heparin therapy than in patients with a history of thrombosis, who were studied more than 3 months after the acute event, and were either on, or had been on, oral anticoagulant therapy. HCII and fibrinogen were significantly correlated in all three groups of patients. These results were strengthened by those of a follow-up study in 23 patients with acute DVT. Changes in plasma HCII activity paralleled those of fibrinogen. This suggests that HCII might behave like an acute phase reactant in patients with thrombosis and that the measurement of its plasma level as a risk factor for thrombosis should be performed some time after the acute episode. In conclusion, the prevalence of HCII deficiency in patients with a history of thrombosis might have been underestimated in series which included patients with acute thrombosis.     

Heparin cofactor II-proteinase reaction products exhibit neutrophil chemoattractant activity

The physiologic function of the plasma glycoprotein heparin cofactor II (HCII) is not well understood. An in vivo role for thrombin (IIa) inhibition by HCII in the presence of certain glycosaminoglycans (dermatan sulfate and heparin) can be proposed. Many proteins, such as complement components, can be proteolyzed to generate secondary bioactive molecules. HCII is a substrate for the human neutrophil (PMN) proteinases cathepsin G (CG) and elastase (LE). We found that degradation of HCII by CG or LE generated products with potent PMN chemotactic activity, which did not stimulate the PMN oxidative burst. Our results suggest that HCII may be a physiologic regulator of the acute inflammatory response.     

Alpha-2-macroglobulin may provide protection from thromboembolic events in antithrombin III-deficient children.

Antithrombin III (ATIII) deficiency has been implicated in adults as a predisposing factor to thrombosis; however, thromboembolic complications are rare in children with the same deficiency. We hypothesized that because of the elevated levels of plasma alpha-2-macroglobulin (alpha 2M) throughout childhood, plasmas of ATIII-deficient children inhibit thrombin more efficiently than those of ATIII-deficient adults. In total, 14 ATIII-deficient adults (ages 25 to 46 years), 13 ATIII-deficient children (ages 2 to 13 years), 9 normal children (ages 3 to 15 years), and 16 normal adults were studied. We measured thrombin inhibition in these plasmas, as well as the contributions of ATIII, alpha 2M, and heparin cofactor II (HCII) as thrombin inhibitors in each plasma. 125I-alpha-thrombin, 25 nmol/L, was added to each plasma (defibrinated with Arvin at 37 degrees C), and 90 seconds later the free thrombin and thrombin-inhibitor complexes were quantitated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric scanning. Plasma from ATIII-deficient adults inhibited significantly less thrombin (12.8 +/- 0.6 nmol/L) than both normal adults (16.1 +/- 0.3 nmol/L, P less than .01), normal children (15.7 +/- 0.4 nmol/L, P less than .01), or ATIII-deficient children (15.5 +/- 0.3 nmol/L, P less than .01). There was no significant difference between the total concentration of thrombin inhibited by ATIII-deficient children and either normal adult or normal children groups. In addition, plasmas of ATIII-deficient children inhibited thrombin significantly more efficiently than plasma of ATIII-deficient adults (P less than .01). In the ATIII-deficient patients there was a significant correlation between the alpha 2M level and ability to inhibit thrombin (P less than .01), but no correlation between either ATIII or HCII levels and thrombin inhibition. On the addition of heparin (0.4 U/mL) to plasma, all four types of plasma inhibited thrombin to the same extent. Although ATIII was the predominant inhibitor in all heparinized plasmas, HCII inhibited more thrombin in the ATIII-deficient patients than in normal patients (2.8 +/- 0.3 v 1.2 +/- 0.2 nmol/L, P less than .01). We hypothesize that the lower risk of thromboembolic complications in ATIII-deficient children may be due in part to the protective effect of elevated alpha 2M levels during childhood.     

Heparin cofactor II modulates the response to vascular injury

Heparin cofactor II (HCII) has several biochemical properties that distinguish it from other serpins: (1) it specifically inhibits thrombin; (2) the mechanism of inhibition involves binding of an acidic domain in HCII to thrombin exosite I; and (3) the rate of inhibition increases dramatically in the presence of dermatan sulfate molecules having specific structures. Human studies suggest that high plasma HCII levels are protective against in-stent restenosis and atherosclerosis. Studies with HCII knockout mice directly support the hypothesis that HCII interacts with dermatan sulfate in the arterial wall after endothelial injury and thereby exerts an antithrombotic effect. In addition, HCII deficiency appears to promote neointima formation and atherogenesis in mice. These results suggest that HCII plays a unique and important role in vascular homeostasis.     

Interaction of protamine sulfate with thrombin

Protamine sulfate (salmine), a basic protein with a molecular weight of 4,626 +/- 109, is a known antiheparin agent which in the absence of heparin demonstrates an anticoagulant activity. To date, much work has been done to elucidate the interaction of heparin with thrombin and its physiologic inhibitor, Antithrombin III (ATIII). Little is known, however, about the mechanism of anticoagulant action of protamine sulfate and its mode of thrombin inactivation. We provide information about the interaction of protamine sulfate with purified, labeled thrombin and ATIII through binding experiments in which protamine is shown to inhibit the inactivation of thrombin by ATIII. Furthermore, we show in clotting assays that protamine sulfate has an inhibitory effect on thrombin in the conversion of fibrinogen to fibrin, and that this inhibition is concentration dependent, partial, and reversible.     

Plasma dermatan sulfate proteoglycan in a patient on chronic hemodialysis

A 68-year-old man on chronic hemodialysis for 6 years, presented with a spontaneous psoas muscle hemorrhage. Investigations showed intermittently elevated activated partial-thromboplastin time and thrombin time. Preliminary investigations suggested a heparin-like inhibitor in the patient's plasma, but no anti-Xa activity could be detected. Investigation of the ability of patient plasma to inhibit exogenous thrombin showed that most thrombin was inhibited by heparin cofactor II, in contrast to normal plasma in which most thrombin was inhibited by antithrombin III. Treatment of plasma with glycosaminoglycan-degrading enzymes suggested the presence of dermatan sulfate (DS) in patient plasma. This was confirmed in a heparin cofactor II-dependent antithrombin assay for DS that showed anticoagulant equivalent to 2.2 +/- 0.3 micrograms/mL (mean +/- SD) of porcine mucosal DS. Of this activity, approximately 90% was sensitive to enzymes that degrade DS. The glycosaminoglycan containing fraction of plasma was isolated and subjected to gel chromatography. Anticoagulant activity eluted from Sephadex G-100 (Pharmacia, Montreal, Quebec, Canada) as two peaks with Kav of 0.10 and 0.45. After treatment with base, the Kav of the higher molecular weight species was increased to 0.55. This activity was completely sensitive to enzymes that degrade DS. Thus, the active DS was present as a proteoglycan. The lower molecular weight material was not sensitive to enzymes that degrade DS or heparan sulfate and it was active in the heparin cofactor II- dependent antithrombin assay but not in an antithrombin III-dependent antithrombin assay. This activity was not degraded by heating. Subsequently, measurement of DS activity was performed in plasmas obtained from eight other patients on hemodialysis before administration of heparin that showed that all patients had DS activity present that varied from 0.05 to 0.4 microgram/mL. No enzyme-resistant activity could be shown in these patients. In summary, a circulating anticoagulant with properties of DS is present in patients requiring hemodialysis.     

Accelerated atherogenesis and neointima formation in heparin cofactor II deficient mice

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin when bound to dermatan sulfate or heparin. HCII-deficient mice are viable and fertile but rapidly develop thrombosis of the carotid artery after endothelial injury. We now report the effects of HCII deficiency on atherogenesis and neointima formation. HCII-null or wild-type mice, both on an apolipoprotein E-null background, were fed an atherogenic diet for 12 weeks. HCII-null mice developed plaque areas in the aortic arch approximately 64% larger than wild-type mice despite having similar plasma lipid and glucose levels. Neointima formation was induced by mechanical dilation of the common carotid artery. Thrombin activity, determined by hirudin binding or chromogenic substrate hydrolysis within 1 hour after injury, was higher in the arterial walls of HCII-null mice than in wild-type mice. After 3 weeks, the median neointimal area was 2- to 3-fold greater in HCII-null than in wild-type mice. Dermatan sulfate administered intravenously within 48 hours after injury inhibited neointima formation in wild-type mice but had no effect in HCII-null mice. Heparin did not inhibit neointima formation. We conclude that HCII deficiency promotes atherogenesis and neointima formation and that treatment with dermatan sulfate reduces neointima formation in an HCII-dependent manner.     

Inhibition of heparin activity in plasma by soluble fibrin: evidence for ternary thrombin-fibrin-heparin complex formation

The ability of heparin to dramatically enhance the inactivation of thrombin (IIa) by antithrombin III (ATIII) in buffer is negated through formation of a IIa-fibrin-heparin ternary complex (Hogg and Jackson, Proc Natl Acad Sci USA 86:3619, 1989; Hogg and Jackson, J Biol Chem 265:241, 1990). IIa, in this ternary complex, is protected from inactivation by ATIII. Our aim was to determine whether fibrin also compromises heparin efficacy in plasma. We found that soluble fibrin ablated the heparin-mediated prolongation of the thrombin time with half-maximal effect at 60 nmol/L fibrin. The heparin-mediated prolongation of the activated partial thromboplastin time (APTT) was also reduced by fibrin with half-maximal effects at 140 nmol/L fibrin using 0.12 U/mL heparin and 500 nmol/L fibrin using 0.25 U/mL heparin. The mechanism of inhibition of heparin activity by fibrin in plasma was determined by measuring IIa-ATIII complexes by enzyme-linked immunosorbent assay (ELISA). Fibrin was found to inhibit the heparin- catalyzed inactivation of IIa by ATIII with half-maximal effect at 97 +/- 19 nmol/L fibrin. Fibrin had no effect on the heparin-catalyzed inactivation of factor Xa by ATIII in plasma, using either standard heparin, a heparinoid preparation (Orgaran; Organon, Lane Cove, Sydney, Australia), or low-molecular weight heparin. These findings imply that fibrin is a potent modulator of heparin activity in vivo by inhibiting heparin-catalyzed IIa-ATIII complex formation through formation of ternary IIa-fibrin-heparin complexes.     

Molecular and cellular basis for type I heparin cofactor II deficiency (heparin cofactor II Awaji)

Heparin cofactor II (HCII) is a serine proteinase inhibitor in human plasma that rapidly inhibits thrombin in the presence of dermatan sulfate or heparin. To understand the molecular mechanism for HCII deficiency in a patient with reduced circulating HCII antigen, we studied a Japanese patient with type I HCII deficiency who suffered from angina pectoris and coronary artery disease. Polymerase chain reaction (PCR)-based sequence analysis showed that the propositus' gene for HCII (HCII Awaji gene) had a thymine insertion after codon (GAT) for Asp88 in exon II, resulting in a frameshift mutation. Consequently, the abnormal HCII Awaji protein was suggested to have an altered amino acid sequence from position 89 and terminate at 107, thus being composed of the NH2-terminal one fifth of normal HCII and dysfunctional for thrombin inhibition. The molecular weight and pI value of HCII Awaji were calculated to be 12,040 and 3.6, respectively, without posttranslational modification. Mutagenic PCR followed by the Tsp509I digestion showed that a half of the PCR products derived from the propositus and his sister was cleaved, suggesting that his sister also has the same mutant allele. Crossed-immunoelectrophoresis and Western blot analyses of plasma and urine from the the propositus and of plasma from his sister did not provide evidence for the existence of the abnormal HCII, suggesting that little truncated HCII was circulating in the patient's blood. However, stable expression assay using human kidney 293 cells transfected with the expression vector containing cDNA encoding wild-type or Awaji-type HCII showed that mutant as well as wild-type HCII was secreted into culture medium normally. These results suggest that the abnormal HCII Awaji protein is secreted normally, but rapidly degraded in the circulating blood.     

Studies on the effect of calcium in interactions between heparin and heparin cofactor II using surface plasmon resonance.

Heparin is the most acidic polysaccharide in the human body and as a result interacts with many cationic species, including ions and proteins, giving rise to myriad biologic activities. Heparin cofactor II (HCII) is a serine protease inhibitor that resembles antithrombin (ATIII) in its ability to be activated by heparin. The interaction of heparin with HCII has been the focus of many studies using affinity chromatography and fluorescence spectroscopy. In this study, surface plasmon resonance (SPR) spectroscopy was used to quantitatively measure the interaction of heparin and HCII using a heparin biochip prepared by covalently immobilizing preformed albumin-heparin conjugate. HCII contains multiple EF hand domains that represent putative calcium ion binding sites. The interactions of HCII with heparin, low-molecular-weight heparin, and heparin oligosaccharides (disaccharide, tetrasaccharide, hexasaccharide) were examined in solution competition experiments using SPR. The results also showed while calcium ions enhanced the heparin/HCII interaction, the activity of heparin-HCII complex against thrombin was not calcium dependent but can be enhanced by the presence of calcium.     

Protein-bound heparin/heparan sulfates in human adult and umbilical cord plasma

We used thrombin times and a competitive radiometric assay to identify, quantitate and characterize endogenous heparin-like molecules in umbilical cord (n = 58) and normal adult (n = 25) plasma. Thrombin times for cord plasma (29.6+/-3.6 s) were significantly longer (p< or = 0.0005) than those for adult plasma (18. 9+/-2.3 s), suggesting increased endogenous heparins. A radiometric assay based on the displacement of (125)I-heparin from protamine-Sepharose revealed that protease-digested plasma contained heparin/heparan sulfate, and plasma that was not digested with protease appeared not to contain heparin/heparan sulfate. More heparin/heparan sulfate was identified in cord than in adult plasma (p< or =0.05), but heparinase digestion produced significantly (p< or =0.001) reduced concentrations of heparin/heparan sulfate in only 39% of the samples. The lack of heparinase sensitivity in 61% of the protease-digested samples apparently was due to low molecular weight (LMW) heparins, for control heparin fragments of 5 kD that did not extend thrombin times were also less affected by heparinase, but the same LMW heparins were detected by radiometric assay. Despite normal thrombin times in all samples, the amounts of endogenous heparin/heparan sulfate identified in protease-digested samples by radiometric assay were of sufficient concentrations to produce inordinately prolonged thrombin times when compared with the same concentrations of unfractionated heparin. Collectively, these findings suggest the presence of a plasma reservoir of endogenous heparin/heparan sulfates in normal cord and adult plasma. These endogenous heparin/heparan sulfates are bound to plasma proteins, and an as yet undetermined proportion of these bound heparin/heparans are most likely LMW molecules.     

Low plasma heparin cofactor II levels in thalassaemia syndromes are corrected by chronic blood transfusion

Low plasma heparin cofactor II (HCII) levels are associated with a thrombotic tendency, and we have previously shown these to be decreased in a variety of haemolytic conditions. The risk of thrombosis is recognized to be increased in both thalassaemia major (TM) and intermedia (TI), although the exact mechanisms are poorly understood.
HCII levels have therefore been compared in 20 untrans-fused patients with TI and 20 regularly transfused TM patients to determine the influence of transfusion on HCII. Additionally, untransfused TI patients have been commenced on regular red cell transfusion and the effects on correction of low HCII levels investigated. HCII levels were significantly lower in the untransfused TI patients (mean 0–56 – 0–06U/ml) compared to TM patients (mean 0–85 – 0-lU/ml; P< O'OOl). Levels in TI were significantly less than in healthy age-matched controls (P < 0–001) and correlated with Hb values (r = 0'8), whereas levels in TM
were at the lower end of the normal range. ATIII values were within the normal reference range in both TI and TM, and HCII antigen showed a parallel reduction to HCII activity, indicating that reduction in HCII is not a consequence of increased thrombin consumption. Three patients with TI were commenced prospectively on hypertransfusion programmes which resulted in a slow normalization of their levels taking 2–3 months.
These findings support a hypothesis that the low HCII levels are related to increased red cell turnover and can be normalized once this turnover has been suppressed by hypertransfusion, The thrombotic risk to patients with low HCII levels in the presence of haemolysis might in principle be decreased by such transfusion regimes.     

Heparan sulfate and dermatan sulfate inhibit the generation of thrombin activity in plasma by complementary pathways

Heparan with a low affinity for antithrombin III has previously been demonstrated to inhibit thrombin generation in both normal plasma and plasma depleted of antithrombin III. In addition, standard heparin and heparin with a low affinity for antithrombin III have been demonstrated to have equivalent inhibitory actions on thrombin generation in plasma depleted of antithrombin III. These observations prompted the investigation of the effects of four normal vessel wall glycosaminoglycans (heparan sulfate, dermatan sulfate, chondroitin-4- sulfate, and chondroitin-6-sulfate) on the intrinsic pathway generation of thrombin and factor Xa and on the inactivation of thrombin and factor Xa in plasma. Heparan sulfate inhibited thrombin generation and accelerated the inactivation of added thrombin and factor Xa in normal plasma but not in antithrombin III-depleted plasma. In contrast, dermatan sulfate inhibited thrombin generation in both normal and antithrombin III-depleted plasma. In addition, heparan sulfate was an effective inhibitor of factor Xa generation, while dermatan sulfate was not. Neither chondroitin-4-sulfate nor chondroitin-6-sulfate inhibited the generation of thrombin or factor Xa nor did they accelerate the inactivation of factor Xa or thrombin by plasma. These results suggest that heparan sulfate acts primarily by potentiating antithrombin III, while dermatan sulfate acts by potentiating heparin cofactor II. The inhibition of thrombin generation by heparan sulfate and dermatan sulfate thus appears to occur by complementary pathways, both of which may contribute to the anticoagulation of blood in vivo.     

Decreased heparin cofactor II activity is associated with impaired endothelial function determined by brachial ultrasonography and predicts cardiovascular events

BackgroundHeparin cofactor II (HCII) could inactivate thrombin after binding to dermatan sulfate at injured arterial walls, and has been shown to be a novel and independent antiatherosclerotic factor. However, the relation between plasma HCII activity and peripheral vascular endothelial function remains unclear.MethodsA total of 199 patients (mean age, 63 ± 14 years) were enrolled and followed up for a median period of 24 months. Endothelial function was assessed using brachial ultrasonography to determine endothelium dependent flow-mediated vasodilation (FMD). Cox regression analyses were conducted for the 199 subjects, with cardiovascular events being defined as myocardial infarction (MI), percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG), ischemic stroke, and peripheral artery revascularization.ResultsA total of 31 patients (16%) had cardiovascular events. Patients with cardiovascular events had significantly lower HCII activity (112 ± 34 versus 127 ± 34%, p = 0.027) and lower antithrombin III (ATIII) activity (82 ± 12 versus 88 ± 13%, p = 0.014) than those without events. By multivariate analysis, age (p = 0.012), hsCRP (p = 0.020) and HCII activity (p = 0.035) were correlated with FMD. Kaplan-Meier analysis was performed and showed plasma HCII (p = 0.036) and ATIII activities (p = 0.005) were predictors of cardiovascular events. By Cox regression analysis, plasma HCII activity (p = 0.026) could be an independent predictor of future cardiovascular events, but not ATIII.ConclusionsThe present study demonstrates that plasma HCII activity is positively correlated with endothelial vasodilator function. Furthermore, plasma HCII activity could be a predictor of future cardiovascular events in patients with suspected coronary artery disease, suggesting its role in atherosclerosis.     

Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions: antithrombin III- and heparin cofactor II-independent inhibition of factor X activation by factor IXa-factor VIIIa complex and heparin cofactor II-dependent inhibition of thrombin

The inhibition mechanism of a polysaccharide anticoagulant, depolymerized holothurian glycosaminoglycan (DHG), was examined by analyzing its effects on the clotting time of human plasma depleted of antithrombin III (ATIII), of heparin cofactor II (HCII), or of both heparin cofactors. The effect exerted by this agent on the activation of prothrombin and factor X in purified human components were also examined and all effects were compared with those of other glycosaminoglycans (GAGs). The capacity of DHG to prolong activated partial thromboplastin time was not reduced in ATIII-depleted, HCII- depleted, HCII-depleted, or ATIII- and HCII-depleted plasma, whereas its capacity to prolong prothrombin time and thrombin clotting time was reduced in HCII-depleted plasma. DHG inhibited the amidolytic activity of thrombin in the presence of HCII with a second order rate constant of 1.2 x 10(8) (mol/L)-1 min-1. These results indicated that DHG has two different inhibitory activities, one being an HCII-dependent thrombin inhibition and the other an ATIII- and HCII-independent inhibition of the coagulation cascade. The heparin cofactors- independent inhibitory activity of DHG was investigated in the activation of prothrombin by factor Xa and in the activation of factor X by tissue factor-factor VIIa complex or by factor IXa. DHG significantly inhibited the activation of factor X by factor IXa in the presence of factor VIIIa, but not in the absence of factor VIIIa. The interaction between DHG and factors IXa, VIIIa, and X was investigated with a DHG-cellulofine column, on which DHG had strong affinity for factors IXa and VIIIa. These findings show that the heparin cofactors- independent inhibition exhibited by DHG was caused by inhibition of the interaction of factor X with the intrinsic factor Xase complex, probably by binding to the factor IXa-factor VIIIa complex.     

Fibrinopeptide A levels indicative of pulmonary vascular thrombosis in patients with primary pulmonary hypertension

Although the mechanisms involved in the pathophysiology of primary pulmonary hypertension have not yet been delineated, thrombosis has been implicated. This study was designed to determine whether thrombin activity as reflected by plasma concentrations of fibrinopeptide A (FPA), a marker of the action of thrombin on fibrinogen, is increased in patients with primary pulmonary hypertension. To evaluate fibrinolytic activity, we measured plasma concentrations of tissue-type plasminogen activator, plasminogen activator inhibitor-1, and cross-linked fibrin degradation products. We studied 31 patients with primary pulmonary hypertension. Plasma FPA concentrations measured by radioimmunoassay, were elevated to 87.4 +/- 36.9 ng/ml (mean +/- SEM). Fifteen minutes after administration of heparin (5,000 U), FPA concentrations decreased to 6.8 +/- 1.4 ng/ml (p less than 0.001 compared with preheparin levels). In 21 of 30 patients (70%), FPA concentrations after heparin administration were less than half the preheparin levels, a response consistent with inhibition of thrombin by heparin and the short half-life of FPA. Despite evidence for marked thrombin activity, plasma concentrations of cross-linked fibrin degradation products were normal in all but four patients. Plasminogen activator inhibitor-1 activity was elevated in 19 of the 27 patients in whom it was measured, potentially limiting the fibrinolytic response. The elevations of FPA indicate that thrombin activity is increased in vivo in patients with primary pulmonary hypertension. Thus, sequential assays of plasma markers of thrombosis and fibrinolysis in vivo may help identify those patients who may benefit from treatment with anticoagulants.     

Molecular and functional characterization of a natural homozygous Arg67His mutation in the prothrombin gene of a patient with a severe procoagulant defect contrasting with a mild hemorrhagic phenotype

In a patient who presented with a severe coagulation deficiency in plasma contrasting with a very mild hemorrhagic diathesis a homozygous Arg67His mutation was identified in the prothrombin gene. Wild-type (factor IIa [FIIa]-WT) and mutant Arg67His thrombin (FIIa-MT67) had similar amidolytic activity. By contrast, the k(cat)/K(m) value of fibrinopeptide A hydrolysis by FIIa-WT and FIIa-MT67 was equal to 2.1 x 10(7) M(-1)s(-1) and 9 x 10(5) M(-1)s(-1). Decreased activation of protein C (PC) correlated with the 33-fold decreased binding affinity for thrombomodulin (TM; K(d) = 65.3 nM vs 2.1 nM, in FIIa-MT67 and in FIIa-WT, respectively). In contrast, hydrolysis of PC in the absence of TM was normal. The Arg67His mutation had a dramatic effect on the cleavage of protease-activated G protein-coupled receptor 1 (PAR-1) 38-60 peptide (k(cat/)K(m) = 4 x 10(7) M(-1)s(-1) to 1.2 x 10(6) M(-1)s(-1)). FIIa-MT67 showed a weaker platelet activating capacity, attributed to a defective PAR-1 interaction, whereas the interaction with glycoprotein Ib was normal. A drastic decrease (up to 500-fold) of the second-order rate constant pertaining to heparin cofactor II (HCII) interaction, especially in the presence of dermatan sulfate, was found for the FIIa-MT67 compared with FIIa-WT, suggesting a severe impairment of thrombin inhibition by HCII in vivo. Finally, the Arg67His mutation was associated with a 5-fold decrease of prothrombin activation by the factor Xa-factor Va complex, perhaps through impairment of the prothrombin-factor Va interaction. These experiments show that the Arg67His substitution affects drastically both the procoagulant and the anticoagulant functions of thrombin as well as its inhibition by HCII. The mild hemorrhagic phenotype might be explained by abnormalities that ultimately counterbalance each other.     

Determination of the levels of unfractionated and low-molecular-weight heparins in plasma: their effect on thrombin-mediated feedback reactions in vivo. Preliminary results after subcutaneous injection.

We define a standard independent unit (SIU) of heparin as that amount that, in plasma containing 1 mumol of ATIII, raises the (pseudo-)first-order breakdown constant of factor Xa by 1 min-1. These units measure all material with a high affinity for ATIII (HAM); only material above the critical chain length of 17 monosaccharide units (above critical chain length material; ACLM) catalyzes the inactivation of thrombin. An SIU of ACLM is therefore analogously defined as the amount that, in plasma containing 1 mumol of ATIII, will raise the (pseudo-)first-order breakdown constant of thrombin by 1 min-1. Of any given heparin preparation one can determine the specific HAM and ACLM activities in terms of SIU/mg. On the basis of the factor Xa and thrombin breakdown constants found in a plasma sample one can then determine the levels of HAM and ACLM. Preliminary experiments were carried out in plasma samples obtained after subcutaneous injection of unfractionated heparin (UFH) and of two types of low-molecular-weight heparin (LMWH). About three times more of UFH activity than of LMWH activity has to be injected to obtain the same levels of ACLM in the plasma. Only with the LMWHs significant amounts of BCLM are found, which rises higher and persists longer than the ACLM. We determined the course of thrombin generation in platelet-rich plasma (PRP) and in platelet-poor plasma (PPP), as well as in the PPP factor Xa generation curve and the course of prothrombin conversion. The observed inhibitions correlated much better with the levels of ACLM than with those of below critical chain length material. The difference between UFH and LMWHs can therefore not be explained in terms of antithrombin and anti-factor-Xa activity. The essential difference between UFH and LMWH appears in the feedback effect of thrombin in PRP, where thrombin generation is both inhibited and retarded by LMWH, while it is only retarded but hardly inhibited by UFH.