George J. Broze

Factor XI activation in a revised model of blood coagulation

Coagulation factor XI is activated in vitro by factor XIIa in the presence of high molecular weight kininogen (HMWK) and a negatively charged surface. Factor XII deficiency is not associated with bleeding, which suggests that another mechanism for factor XI activation exists in vivo. A revised model of coagulation is proposed in which factor XI is activated by thrombin. In the absence of cofactors, thrombin is more effective (kcat/Km = 1.6 x 10(5)) than factor XIIa (1.7 x 10(4)) in activating factor XI. Dextran sulfate enhances activation of factor XI by thrombin 2000-fold; part of this effect is due to autoactivation of factor XI by activated factor XI.

Absence of thrombosis in subjects with heterozygous protein C deficiency.

Protein C deficiency has been thought to be associated with an increased risk of venous thrombosis. To establish a normal range of values, we used a two-site monoclonal-antibody assay to measure protein C levels in 699 healthy adults. The distribution was log normal; 95 percent of the values ranged from 70 to 140 percent of the overall mean (4.03 micrograms per milliliter). Two subjects had levels more than 3.5 SD below the mean (34 and 50 percent), consistent with heterozygous deficiency. We also screened 4723 other blood donors and found 8 additional unrelated subjects with levels from 33 to 51 percent of normal. Autosomal inheritance of heterozygous protein C deficiency was confirmed in them by a detailed study of four families. Levels from 55 to 65 percent of normal are consistent with either heterozygous deficiency or the lower end of the distribution of normal values, and were found in 79 of 5422 subjects when the two groups were combined. None of the subjects had any history of venous thrombosis. We conclude that heterozygous deficiency of protein C has a prevalence of 1 in 200 to 300, a figure consistent with the known number of homozygous infants recently identified, and that levels consistent with heterozygous deficiency are found in 1 in 60 healthy adults but are not detectably associated with a risk of thrombosis.

Tissue factor pathway inhibitor and the current concept of blood coagulation.

&NA; Tissue factor pathway inhibitor (TFPI) is a multivalent, Kunitz‐type plasma proteinase inhibitor that regulates tissue factor‐induced coagulation. TFPI directly inhibits activated factor X and, in a factor Xa‐dependent manner, produces feedback inhibition of the factor VIIa/tissue factor catalytic complex. The properties of this inhibitor help explain the clinical need for ‘extrinsic’ and ‘intrinsic’ coagulation pathways and have led to a reformulation of the coagulation cascade. In the revised hypothesis, factor VIIa/tissue factor is responsible for the initiation of coagulation but, owing to TFPI‐mediated feedback inhibition, amplification of the procoagulant response though the actions of factor VIII, IX and XI is required for sustained haemostasis.

Thrombin-activatable fibrinolysis inhibitor (TAFI) deficiency is compatible with murine life

To investigate the consequence of deficiency in thrombin-activatable fibrinolysis inhibitor (TAFI), we generated homozygous TAFI-deficient mice by targeted gene disruption. Intercrossing of heterozygous TAFI mice produced offspring in the expected Mendelian ratio, indicating that transmission of the mutant TAFI allele did not lead to embryonic lethality. TAFI-deficient mice developed normally, reached adulthood, and were fertile. No gross physical abnormalities were observed up to 24 months of age. Hematological analysis of TAFI-deficient mice did not show any major differences including plasma fibrinogen level, prothrombin time, and activated partial thromboplastin time. TAFI-deficient mice did not suffer from excess bleeding as determined by blood loss following tail transection, although their plasma failed to prolong clot lysis time in vitro. In vivo, TAFI deficiency did not influence occlusion time in either an arterial or a venous injury model. TAFI deficiency did not improve survival rate compared with the wild-type in thrombin-induced thromboembolism, factor X coagulant protein-induced thrombosis, and endotoxin-induced disseminated intravascular coagulation. Furthermore, TAFI deficiency did not alter kaolin-induced writhing response, implying that TAFI does not play a major role in bradykinin catabolism. The current study demonstrates that TAFI deficiency does not change normal responses to acute challenges.

A murine model of factor XI deficiency.

To facilitate investigations into the physiologic and pathologic roles of factor XI, we have developed a murine model of severe factor XI deficiency using the technique of homologous recombination in embryonic stem cells. The factor XI gene was disrupted by introducing a neomycin phosphotransferase gene into the fifth exon. The activated partial thromboplastin times of homozygous null mice were prolonged (158- > 200 s) compared with wild type (25-34 s) and heterozygous null (40-61 s) litter mates. Factor XI activity was absent from the plasma of mice homozygous for the null mutation and factor XI mRNA was undetectable by Northern blot and reverse transcription/PCR in the livers of homozygous null animals. The genotypes of progeny from matings of mice heterozygous for the factor XI null allele followed the expected Mendelian ratio (1:2:1, wild type 26%, heterozygote null 54%, homozygous null 20%), indicating that severe factor XI deficiency did not result in increased intrauterine death. Results of a tail transection bleeding time assay were similar for wild type and homozygous null animals with, at most, a tendency for slightly prolonged bleeding in the homozygous null animals. The factor XI deficient mice are a unique tool for evaluating the role of factor XI in normal hemostasis and pathologic coagulation.

Human Protein Z.

Protein Z was purified from human plasma by a four-step procedure which included barium citrate adsorption, ammonium sulfate fractionation, DEAE-Sepharose chromatography, and blue agarose chromatography with a yield of 20%. It is a 62,000 mol wt protein with an extinction coefficient of 12.0. The concentration of Protein Z in pooled, citrated plasma is 2.2 micrograms/ml and its half-life in patients starting warfarin anticoagulation therapy is estimated to be less than 2.5 d. The NH2-terminal sequence is Ala-Gly-Ser-Tyr-Leu-Leu-(Gla)-(Gla)-Leu-Phe-(Gla)-Gly-Asn-Leu. Neither Protein Z nor its cleavage products, which were obtained by treatment of Protein Z with thrombin or plasmin, incorporated [3H]diisopropyl fluorophosphate. The physiological function of Protein Z remains unknown.

Binding of human factor VII and VIIa to monocytes.

Human coagulation Factors VII and VIIa bind with equal affinity to monocytes stimulated with endotoxin. Equilibrium binding studies performed at 0 degrees C using 125I-labeled Factor VII and VIIa showed the dissociation constant (Kd) to be congruent to 82 pM with congruent to 3,600 binding sites/monocyte. Ca++ was required for Factor VII and VIIa interaction with monocytes (optimal CaC12 concentration greater than or equal to 2.5 mM) and binding was reversed by the addition of EDTA. The rate of conversion of Factor X to Xa in mixtures containing Factor VIIa and monocytes was directly related to the quantity of Factor VIIa bound to the monocyte surface. Thus the monocyte binding sites appear to represent tissue factor. Competition experiments showed that Factor VII and VIIa bind to the same monocyte sites and further, that unlabeled Factor VII and VIIa have the same affinity for the binding sites as the 125I-labeled proteins.

Glycosyl phosphatidylinositol anchorage of tissue factor pathway inhibitor

Background—The endothelium is a major source of tissue factor pathway inhibitor (TFPI), the endogenous regulator of TF-induced coagulation, and a significant proportion of the expressed TFPI remains associated with the endothelial surface. Methods and Results—Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment reduced TFPI at the surface of cultured endothelial cells by ≈80%, and at least a portion of the TFPI released by PI-PLC contained an intrinsic glycosylphosphatidylinositol (GPI) anchor that is recognized by anti-crossreactive determinant antibodies. Endothelial cells express both of the alternatively spliced forms of TFPI mRNA at a ratio of TFPI&bgr;/TFPI&agr; mRNA of ≈0.1 to 0.2. In Chinese hamster ovary (CHO) cells, TFPI&agr; is predominantly secreted, whereas TFPI&bgr; is a GPI-anchored membrane protein. Like TFPI&bgr;, the small proportion of the TFPI&agr; expressed by CHO cells that remains surface associated is also released by PI-PLC treatment, suggesting that it is bound to a separate GPI-anchored protein(s) at the surface of the cells. Conclusions—Both direct (TFPI&bgr;) and indirect (TFPI&agr;) GPI-mediated membrane anchorage is involved in localizing TFPI to the surface of cells.

Heritability of plasma concentrations of clotting factors and measures of a prethrombotic state in a protein C-deficient family

Summary.  Background: Earlier studies found strong support for a genetic basis for regulation of coagulation factor levels and measures of a prethrombotic state (d‐dimer, prothrombin fragment 1.2). Objectives: Estimation of how much of the variation in the levels of coagulation factors and measures of a prethrombotic state, including measures of protein C activation and inactivation, could be attributed to heritability and household effect. Patients and methods: Blood samples were collected from 330 members of a large kindred of French‐Canadian origin with type I protein C deficiency. Heritability and common household effect were estimated for plasma concentrations of prothrombin, factor (F)V, factor VIII, factor (F)IX, fibrinogen, von Willebrand factor (VWF), antithrombin, protein C, protein S, protein Z, protein Z‐dependent protease inhibitor (ZPI), fibrinopeptide A (FPA), protein C activation peptide (PCP), activated protein C–protein C inhibitor complex (APC–PCI), activated protein C–α1‐antitrypsin complex (APC–α1AT), prothrombin fragment 1.2 (F1.2) and d‐dimer, using the variance component method in sequential oligo‐genic linkage analysis routines (SOLAR). Results: The highest heritability was found for measures of thrombin activity (PCP and FPA). High estimates were also found for prothrombin, FV, FIX, protein C, protein Z, ZPI, APC–PCI and APC–α1AT. An important influence of shared household effect on phenotypic variation was found for VWF, antithrombin, protein S and F1.2. Conclusions: We found strong evidence for the heritability of single coagulation factors and measures of a prethrombotic state. Hemostatic markers with statistically significant heritability constitute potential targets for the identification of novel genes involved in the control of quantitative trait loci.

Contribution of Host-Derived Tissue Factor to Tumor Neovascularization

Objective—The role of host-derived tissue factor (TF) in tumor growth, angiogenesis, and metastasis has hitherto been unclear and was investigated in this study. Methods and Results—We compared tumor growth, vascularity, and responses to cyclophosphamide (CTX) of tumors in wild-type (wt) mice, or in animals with TF levels reduced by 99% (low-TF mice). Global growth rate of 3 different types of transplantable tumors (LLC, B16F1, and ES teratoma) or metastasis were unchanged in low-TF mice. However, several unexpected tumor/context-specific alterations were observed in these mice, including: (1) reduced tumor blood vessel size in B16F1 tumors; (2) larger spleen size and greater tolerance to CTX toxicity in the LLC model; (3) aborted tumor growth after inoculation of TF-deficient tumor cells (ES TF−/−) in low-TF mice. TF-deficient tumor cells grew readily in mice with normal TF levels and attracted exclusively host-related blood vessels (without vasculogenic mimicry). We postulate that this complementarity may result from tumor-vascular transfer of TF-containing microvesicles, as we observed such transfer using human cancer cells (A431) and mouse endothelial cells, both in vitro and in vivo. Conclusions—Our study points to an important but context-dependent role of host TF in tumor formation, angiogenesis and therapy.