Inhibitory effects of lysine analogues on t-PA induced whole blood clot lysis

The lysine analogues epsilon-aminocaproic acid (EACA) and trans-4-amino-methyl cyclohexane carboxylic acid (AMCA) are used to prevent excessive bleeding in patients with coagulopathies, such as hemophilia and thrombocytopenia, or in those who have received tissue plasminogen activator (t-PA). However, their relative efficacy in inhibiting lysis of clots that have been formed in the presence of exogenous t-PA or that have been formed and then exposed to exogenous t-PA has not been well characterized. The present study utilized blood from normal volunteers and ¹²⁵I-fibrinogen in a dilute whole blood clot assay to determine the relative concentrations of lysine analogues required for inhibition of clot lysis induced by exogenous t-PA. AMCA (0.06 mM) and EACA (0.6 mM) were effective in prolonging clot lysis if (1) whole blood clots were formed and then exposed to a lysine analogue and exogenous t-PA or if (2) whole blood clots were formed in the presence of exogenous t-PA and a lysine analogue. However, their inhibitory effect was markedly reduced if clots were formed in the presence of t-PA and then exposed to either of the lysine analogues. The analogues did not inhibit the initial binding of t-PA to fibrin. They did inhibit binding of plasminogen to fibrin as well as the activation of plasminogen by t-PA in the absence of fibrin. The data suggest that lysine analogues, even at low concentrations, reduce the rate of t-PA induced whole blood clot lysis by several mechanisms.     

Inhibition of activated protein C by benzamidine derivatives

In studies on the inhibition of activated protein C (APC) by benzamidine derivatives potent inhibitors of APC were found among anilides of 4-amidinophenyl--aminobutyric acid (K_i = 0.58 μmol/1). Several bis-benzamidine derivatives containing a cycloalkanone linking bridge inhibit APC with K_i values near the micromolar range.

Potent and selective inhibitors of thrombin derived from 3- and 4-amidinophenylalanine do not inhibit APC. This is of great importance for further development of these inhibitors as potential anticoagulant drugs.     

Inhibitory effect of activated protein C on platelet aggregation induced by the prothrombin-converting reaction

The present study was undertaken to elucidate the effect on platelet aggregation of the prothrombin-converting reaction on platelets with or without activated protein C (APC). A reaction mixture of washed platelets from human individuals, Factor Xa and prothrombin markedly induced platelet aggregation; maximum aggregation rates, 31.3–92.5%, and times to reach to maximum aggregation, 11.6 to 20.1 min. This aggregation was inhibited by the addition of APC with 50% inhibition concentration (IC₅₀) value of 14.4 U/ml. APC also inhibited thrombin generation in the reaction mixture in a dose-dependent manner with IC50 value of 0.96 U/ml. However, APC did not inhibit the thrombin (0.1 CU/ml)-induced platelet aggregation at concentrations of up to 30 U/ml. These findings suggest that APC has no direct inhibitory effect on platelet aggregation and that APC inhibits platelet aggregation through inhibition of thrombin generation.     

A direct-acting fibrinolytic enzyme from the venom of Agkistrodon contortrix contortrix: effects on various components of the human blood coagulation and fibrinolysis systems

A direct acting fibrinolytic enzyme (fibrolase) has been isolated from venom of the southern copperhead snake (Agkistrodon contortrix contortrix). Time-course experiments established that the venom enzyme cleaves primarily the A-chain of human fibrinogen and fibrin between the Lys-413 and Leu-414 position. The Bβ-chain is cleaved more slowly, while the γ-chain is minimally affected. The cleavage pattern of fibrinogen and fibrin clearly varies from plasmin cleavage of the same molecules. The enzyme does not activate plasminogen or protein C and it is thus different from “Protac”, a protein C activator isolated from the same venom.     

Regulation of endothelial cell protein c activation and fibrinolysis by procoagulant albumin

Endothelial cell regulation of protein C activation and fibrinolysis are important components of the hemostatic response to vascular injury or perturbation. Procoagulant albumin (P-A1), a constituent of normal human plasma has been purified and identified as an inducer of endothelial cell tissue factor activity. The purpose of the studies reported herein was to investigate the effects of P-A1 on human endothelial cell protein C activation and fibrinolysis. P-A1 suppressed protein C activation, enhanced release of plasminogen activator inhibitor-1, but had no effect on tissue-plasminogen activator release. Plasminogen activator inhibitor-1 released by P-A1 was functional as evidenced by the capacity to form a covalent complex with ¹²⁵I-urokinase. Inactive albumin (isolated during the same purification procedure as P-A1, but without tissue factor-inducing activity) did not suppress protein C activation or increase plasminogen activator inhibitor-1 release. These results indicate that P-A1, a component of human plasma, can modulate multiple vascular hemostatic properties, and support the hypothesis that P-A1 is involved in normal or pathologic hemostasis.     

Determination of functional and antigenic protein C inhibitor and its complexes with activated protein C in plasma by ELISA''s

Enzyme-linked immunosorbent assays (ELISA's) were developed for the measurement of protein C inhibitor (PCI) antigen and activity and for its complexes with activated protein C (APC) in plasma. For PCI activity and antigen, APC or anti-PCI, respectively, was immobilized to microtiter plates and PCI bound was detected with labelled anti-PCI antibodíes. For APC:PCI complexes, two different antibodies directed against protein C and PCI were used. The assays for PCI were calibrated with pooled normal human plasma (NHP) and with purified PCI, and for APC:PCI complexes with known concentrations of purified pre-formed complexes added to buffer or to plasma. The lower limit of sensitivity of the PCI activity and antigen assays was 10 ng/ml and 0.5 ng/ml, respectively and for plasma APC:PCI complexes 12 ng/ml. Mean coefficients of variation of 1.5 % to 5.8 % (intro-assay) and 2.1 % to 9.8 % (inter-assay) were found for the assays. For PCI antigen, a range of 56 % to 162 % of the NHP value was obtained in samples from 70 healthy donors (mean ±SD = 98.6 % ±23.1%). For PCI activity, the range was 59 % to 148 % (94.3 % ± 20.2). A good correlation (0.92) was obtained when both assays were compared. Plasma levels of APC:PCI complexes in 30 normals were under the detection limit (< 12 ng/ml). In plasma samples from 10 patients with disseminated intravascular coagulation (DIC) PCI antigen concentrations were decreased (55.6% ±20%) and 8 of the patients had APC-PCI complex levels between 32 and 240 ng/ml (median, 35 ng/ml). After addition of 20 ωg/ml APC to NHP or to protein C depleted plasma, 6.1 μg/ml complexes were recovered after 90 min incubation. Incubation of 10 μg/ml APC with NHP in the presence of 10 U/ml heparin yielded 11 μg/ml complexes after 90 min, which represent more than 90 % of the maximum possible value. Thus, the method should be adequate to study complexes of APC in vivo in clinical conditions in which activation of protein C pathway may occur.     

A case report of deficiency in an inhibitor of calcium-dependent association of protein S with C4B-binding protein suggested by a modified crossed immunoelectrophoresis

We have experienced a coagulation factor VIII-deficient patient whose plasma has normal protein S (PS) activity and masses of free PS and its bound form in complex with C4b-binding protein (C4BP). Although the patient's plasma showed a normal ratio of free PS to PS-C4BP complex in the presence of 5 mM EDTA, the plasma gave an abnormally retarding major C4BP peak together with a major PS peak in the crossed immunoelectrophoresis (CIE) in the presence of 2 mM CaCl₂. It was revealed that the major peak was formed by a mixture of PS-C4BP complex and free form. The addition of normal human plasma (NHP) to the patient's plasma inhibited the retardation of the major PS-C4BP complex. These suggest that the patient's plasma lacks some component(s) to inhibit Ca²⁺-dependent association of PS with C4BP.     

A new function for activated protein C: activated protein C prevents inhibition of plasminogen activators by releasate from mononuclear leukocytes--platelet suspensions stimulated by phorbol diester

Mononuclear leukocytes release an inhibitor of plasminogen activators. Mononuclear leukocyte mixtures (400 to 1,000/mm3) lysed fibrin (8.3 microM) clots in the presence of plasminogen (0.58 microM). Anti-urokinase IgG (0.16 microM) inhibited this fibrinolysis. 2-Deoxyglucose (5 mM) and oligomycin (2.3 microM) also inhibited fibrinolysis. Incubation of mononuclear leukocytes (3,200/microliter) with phorbol-12 myristate 13-acetate (20 nM) for ten minutes at 37 degrees C aggregated the monocyte and platelet components and inhibited fibrinolysis. The releasate from these stimulated cells in dilutions ranging from undiluted to 1:16 inhibited urokinase (1.6 pM) and tissue plasminogen activator (1.4 pM). This releasate did not inhibit plasmin (2.5 nM). Incubation of this releasate with activated protein C (33 nM to 333 nM) for ten minutes at 37 degrees C before addition of either urokinase, or tissue plasminogen activator and plasminogen completely prevented this inhibition. Thrombin, factor Xa, DIP-activated protein C had no affect on this inhibition. We conclude that activated protein C facilitates fibrinolysis by preventing inhibition of plasminogen activators. This may be a mechanism by which activated protein C increases fibrinolytic activity in vivo.     

Fibrin clot properties in women heterozygous for factor V Leiden mutation: Effects of oral contraceptives

Introduction

Oral contraceptives (OC) in the presence of factor V Leiden mutation (FVL) markedly increase the risk of venous thromboembolism (VTE). Little is known about the OC and FVL-related alterations in fibrin clot properties.

Subjects and Methods

Plasma fibrin clot permeability (K_s) and efficiency of lysis, reflected by clot lysis time (CLT) and the rate of D-dimer release from clots (D-D_rate) induced by recombinant tissue plasminogen activator (tPA) were determined in 25 women with a family history of VTE who were heterozygous for FVL [FVL(+/−) - twice, on third-generation OC and after their discontinuation. Female non-carriers of FVL, matched for demographics, using OC and after their discontinuation served as controls (n = 25). All participants had no personal history of VTE.

Results

OC discontinuation in FVL(+/−) women resulted in shortened CLT (− 9%), and increased K_s (+ 4%) and D-D_rate (+ 1.4%; all p < 0.01). Alterations in fibrin clot properties were associated with decreased prothrombin fragments 1 + 2 (F1 + 2) (− 8%), plasminogen activator inhibitor-1 (PAI-1) antigen (− 11%), and thrombin activatable fibrinolysis inhibitor (TAFI) activity (− 20%; all p < 0.01). During OC use FVL(+/−) carriers compared with non-carriers had higher platelet count, activity of PAI-1, TAFI, and tPA, as well as prolonged CLT and higher D-D_max, along with lower D-D_rate and K_s. Multiple regression analysis adjusted for fibrinogen and age, showed that PAI-1 antigen and TAFI activity independently predicted CLT in FVL(+/−) women on OC.

Conclusion

FVL(+/−) is associated with hypofibrinolysis in apparently healthy women and third-generation OC administration unfavorably alters plasma clot characteristics in female FVL(+/−) carriers with a family history of thrombotic events.     

Reduced plasma fibrin clot permeability and susceptibility to fibrinolysis are associated with increased intima-media thickness in patients with primary antiphospholipid syndrome

Introduction

Formation of denser fibrin networks displaying impaired lysability has been reported in subjects at an increased risk of atherosclerosis. Given recent data on prothrombotic fibrin clot phenotype reported in patients with antiphospholipid syndrome (APS), we tested the hypothesis that altered fibrin clot properties are associated with increased intima-media thickness (IMT) observed in PAPS.

Materials and methods

We studied 30 consecutive patients with PAPS and 30 controls matched for age, sex and the type of previous thromboembolism. We assessed plasma fibrin clot permeability (K_s) and clot lysis time (CLT) with their potential determinants. The IMT was measured in 3 segments of the carotid arteries.

Results

Patients with APS had 15.2% lower K_s (p = 0.002) and 9.7% prolonged CLT (p = 0.039) compared with controls. The IMT in the APS group was greater in the common carotid artery (5.7%; p = 0.002), at the bifurcation (17.46%; p < 0.001), and the internal artery (9.26%; p = 0.015). Patients with triple positivity in the antiphospholipid antibody profile (n = 9; 30%) had lower K_s and greater IMT (both, p < 0.05), compared with those with single positivity (n = 13; 43.3%). Multivariate analysis adjusted for potential confounders showed that in APS patients, oxidized low-density lipoproteins (p = 0.019) were the only independent predictor of K_s, while thrombin activatable fibrinolysis inhibitor activity (p < 0.001) predicted CLT. Plasminogen activator inhibitor-1 (PAI-1) was found to be the independent predictor of the IMT in the common carotid artery (p = 0.004), and in the internal carotid artery (p < 0.001).

Conclusions

Reduced K_s and susceptibility to lysis are associated with greater IMT in PAPS, which might contribute to the early atherosclerosis in this disease.     

Hemostatically distinct FFPs equally improve abnormal TEG variables in an in vitro dilutional coagulopathy model

Introduction

To improve fresh frozen plasma (FFP) availability, thawed plasma is stored at 4 °C for up to 5 days and considered equivalent to freshly thawed FFP. However, we have shown that hemostatic potential of thawed plasma is highly variable between donors and significantly diminished during storage. We hypothesized that smaller volumes of plasma with higher hemostatic potential (FFP-H) would be needed to restore normal thrombelastogram (TEG) values compared to plasma with lower hemostatic potential (FFP-L).

Materials and Methods

A dilutional coagulopathy model was established from whole blood by diluting plasma with saline to 23%, while cellular components were kept unchanged. Saline was gradually replaced with equal volumes of FFPs with distinctive hemostatic potentials, which was evaluated by the calibrated automated thrombogram. Clot formation in the presence of tissue factor was evaluated by TEG at baseline and after addition of increasing concentrations of FFP-H and FFP-L.

Results

Blood dilution with saline in the presence of tissue factor resulted in abnormal TEGs that resemble a pattern observed in severely bleeding trauma patients. All FFPs produced similar improvements in TEG variables despite different hemostatic potentials. TEG changes were solely dependent on FFP volume and reached the normal reference range when plasma concentration increased to 40%.

Conclusion

Plasma dilution and tissue factor in whole blood results in an abnormal TEG with a hyperfibrinolytic pattern. A plasma concentration of at least 40% was necessary for TEG normalization after dilution with saline. An effect of FFPs’ hemostatic potential on clot formation could not be detected by TEG in this in vitro model.     

Thrombomodulin alfa treatment in patients with acute promyelocytic leukemia and disseminated intravascular coagulation: A retrospective analysis of an open-label,multicenter, post-marketing surveillance study cohort

Introduction

Patients with acute promyelocytic leukemia (APL) can develop disseminated intravascular coagulation (DIC) that results in life-threatening hemorrhagic complications. Studies regarding the safety and efficacy of thrombomodulin alfa (TM-α; recombinant human soluble thrombomodulin) in patients with APL and DIC are limited.

Materials and methods

A retrospective evaluation was performed on a cohort of 172 patients with APL from an open-label, multicenter, post-marketing surveillance study of TM-α.

Results

Of the 172 patients, 31 were relapse/refractory APL patients, and 141 were newly diagnosed APL patients. Within the first 30 days, 24 patients (14.0%) died, and six of those deaths (3.5%) were due to hemorrhage. In total, 12 patients (7.0%) had severe hemorrhagic complications. Both the early death rate due to hemorrhage as well as the severe hemorrhage rate did not exceed those in some recent population-based studies of patients with APL. Forty-nine patients received TM-α prior to the initiation of antileukemic treatment, and one patient experienced hemorrhagic early death (ED), suggesting that early TM-α treatment appeared to result in a reduction in the hemorrhagic ED rate. Moreover, TM-α improved coagulopathy regardless of concomitant all-trans retinoic acid treatment.

Conclusions

This study confirmed the safety and efficacy of TM-α in daily clinical practice for patients with APL and DIC. TM-α appeared to reduce hemorrhagic early deaths due to DIC in patients with APL who were receiving antileukemic treatment.     

Global haemostasis assays, from bench to bedside

Bleeding and thrombosis are the ultimate clinical outcomes of aberrations in the haemostatic process. Haemostasis prevents excessive blood loss due to the effort of various compartments like the vasculature, blood cells, coagulation and fibrinolysis. The complexity of all processes involved makes the diagnosis of aberrations difficult, cumbersome and expensive. A single assay to detect any factor disturbing this haemostatic balance with high sensitivity and specificity would be of great value, especially if the outcome of this assay correlates well with clinical outcome. Despite years of research, such an assay is not yet available; however, some interesting candidates are under development and combine the effects of various compartments. This review describes the development of global haemostasis assays and summarizes the current state of the art of these haemostasis assays covering thrombin and plasmin generation, turbidity and thromboelastography/thromboelastometry. Finally, we discuss the applicability of global assays in clinical practice and we provide a future perspective on the ongoing development of automation and miniaturisation as it is our belief that these developments will benefit the standardization of global haemostasis assays.