Roles and regulation of phospholipid scramblases.

Phospholipid scramblase activity is involved in the collapse of phospholipid (PL) asymmetry at the plasma membrane leading to externalization of phosphatidylserine. This activity is crucial for initiation of the blood coagulation cascade and for recognition/elimination of apoptotic cells by macrophages. Efforts to identify gene products associated with this activity led to the characterization of PL scramblase (PLSCR) and XKR family members which contribute to phosphatidylserine exposure in response to apoptotic stimuli. Meanwhile, TMEM16 family members were identified to externalize phosphatidylserine in response to elevated calcium in Scott syndrome platelets, which is critical for activation of the coagulation cascade. Herein, we report their mechanisms of gene regulation, molecular functions independent of their scrambling activity, and their potential roles in pathogenic conditions.

JAK2V617F-positive endothelial cells contribute to clotting abnormalities in myeloproliferative neoplasms.

The Janus kinase 2 (JAK2) V617F mutation is the primary pathogenic mutation in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombohemorrhagic incidents are the most common causes of morbidity and mortality in patients with MPNs, the events causing these clotting abnormalities remain unclear. To identify the cells responsible for the dysfunctional hemostasis, we used transgenic mice expressing JAK2V617F in specific lineages involved in thrombosis and hemostasis. When JAK2V617F was expressed in both hematopoietic and endothelial cells (ECs), the mice developed a significant MPN, characterized by thrombocytosis, neutrophilia, and splenomegaly. However, despite having significantly higher platelet counts than controls, these mice showed severely attenuated thrombosis following injury. Interestingly, platelet activation and aggregation in response to agonists was unaltered by JAK2V617F expression. Subsequent bone marrow transplants revealed the contribution of both endothelial and hematopoietic compartments to the attenuated thrombosis. Furthermore, we identified a potential mechanism for this phenotype through JAK2V617F-regulated inhibition of von Willebrand factor (VWF) function and/or secretion. JAK2V617F(+) mice display a condition similar to acquired von Willebrand syndrome, exhibiting significantly less high molecular weight VWF and reduced agglutination to ristocetin. These findings greatly advance our understanding of thrombohemorrhagic events in MPNs and highlight the critical role of ECs in the pathology of hematopoietic malignancies.

Prostate-specific antigen, prostate cancer, and disorders of hemostasis.

Prostate cancer is the most prevalent malignancy in men and the third leading cause of cancer deaths worldwide. Disorders of hemostasis are commonplace in patients with prostate cancer and include disseminated intravascular coagulation, venous thromboembolism, acute coronary syndrome, and postsurgical bleeding. These hemostatic disorders contribute to the mortality and morbidity of prostate cancer. The leading mechanisms proposed to underlie prostate cancer-related coagulopathies are thought to be a hyperexpression of tissue factor, cancer procoagulant, and platelet-activating factor, which is then accompanied by release of large amounts of both prothrombotic and profibrinolytic substances into the bloodstream. Given the generally accepted notion that prostate-specific antigen (PSA) represents an important biomarker in prostate cancer diagnostics, large population screenings were initiated for early detection of cancer. However, recent clinical and economic drawbacks have been recently raised, including evidence that screening exposes patients to a significant risk of both overdiagnosis and overtreatment. Nevertheless, several lines of evidence suggest that PSA may have tumor-suppressing activities. Despite being a member of the vast kallikrein family, which actively interplays with the coagulation cascade, the role of PSA in the pathogenesis of hemostatic disorders observed in prostate cancer patients remains circumstantial and speculative. However, observations that the levels of this cancer marker tend to correlate positively with those of several markers of thrombin generation, and with postsurgical bleeding as well as with coronary atherosclerosis and negative outcomes of myocardial infarction, raise a new and intriguing scenario regarding the pathophysiological role of this serine protease.

Expression of proteins controlling transbilayer movement of plasma membrane phospholipids in the B lymphocytes from a patient with Scott syndrome.

Scott syndrome is a rare inherited bleeding disorder in which platelets and other blood cells fail to promote normal assembly of the membrane-stabilized proteases of the plasma coagulation system. The defect in Scott blood cells is known to reflect inability to mobilize phosphatidylserine from inner plasma membrane leaflet to the cell surface in response to an elevation of Ca2+ at the endofacial surface. To gain insight into the molecular basis of this membrane defect, we examined the expression in Scott cells of plasma membrane proteins that have been implicated to participate in the accelerated transbilayer movement of plasma membrane PL. By both reverse transcriptase-polymerase chain reaction (RT-PCR) and functional assay, the level of expression of the multidrug resistance (MDR)1 and MDR3 P-glycoproteins in immortalized B-lymphoblast cell lines from the patient with Scott syndrome were indistinguishable from matched cell lines derived from normal controls. Whereas the plasma membrane of Scott cells are insensitive to activation of the plasma membrane PL scramblase pathway, it had been shown that PL scramblase protein isolated from detergent-solubilized Scott erythrocytes exhibits normal function when incorporated into proteoliposomes (Stout JG, Basse F, Luhm RA, Weiss HJ, Wiedmer T, Sims PJ: J Clin Invest 99:2232, 1997). Consistent with this finding in Scott erythrocytes, we found that Scott lymphoblasts expressed normal levels of PL scramblase mRNA and protein, and that the deduced sequence of PL scramblase in Scott cells is identical to that of normal controls. These data suggest that the defect in Scott syndrome is related either to aberrant posttranslational processing of the PL scramblase polypeptide or to a defect or deficiency in an unknown cofactor that is required for normal expression of plasma membrane PL scramblase function in situ, or alternatively, reflects the presence of a detergent-dissociable inhibitor of this pathway.

Hypercoagulability and hyperfibrinolysis in patients with melanoma.

The purpose of this study was to evaluate whether or not, using sensitive analytical methods for the measurement of coagulation and fibrinolysis enzyme activity, there was a hypercoagulable state in patients with melanoma, and whether differences existed between those with or without metastases. Seventy-one patients were studied, 45 with localized tumors (stages Ia and Ib) and 26 with metastases (stages II-IV). Plasma level of activated factor VII, prothrombin fragment 1 + 2, thrombin-antithrombin complex, fibrinopeptide A, plasmin-antiplasmin complex and D-dimer were much higher in the whole group of 71 patients than in 45 controls with benign nevi. However, when melanoma patients with or without metastases were compared, there were smaller differences, with only thrombin-antithrombin complex, plasmin-antiplasmin and D-dimer significantly higher in metastatic melanoma. These results indicate that in patients carrying a tumor endowed with high procoagulant activity in vitro, there is a laboratory picture of hypercoagulability with secondary hyperfibrinolysis in vivo. However, differences between patients with localized and metastatic tumors for markers of hypercoagulability are not striking, in spite of the fact that metastatic cells support greater coagulant activity than primary cells in vitro.

Bleeding disorder due to platelet prostaglandin H synthase-1 (PGHS-1) deficiency.

Defective platelet prostaglandin H synthase (PGHS) activity has been recognized as a cause of bleeding disorders, but the defect has not been characterized. We evaluated three female patients aged 37, 48 and 55 who presented with a mild bleeding disorder due to platelet dysfunction. None of the patients had underlying diseases or reported use of aspirin or other nonsteroidal anti-inflammatory drugs. Coagulation screening tests and platelet count were normal in each patient. Platelet aggregation in response to adenosine diphosphate (ADP), collagen and epinephrine were subnormal, characterized by an abnormal second-wave aggregation and propensity for disaggregation. Arachidonate-induced platelet aggregation was defective, whereas PGH2-induced aggregation was normal. Platelet thromboxane A2 (TXA2) production in response to arachidonic acid was reduced in all three patients, i.e. 11.7, 4.6 and 4.4 ng TXB2/3 x 10(8) plt respectively (normal range was 49-81 ng/3 x 10(8) plt), whereas they were normal in response to exogenous PGH2, i.e. 71.4, 56.6 and 48.9 ng/3 x 10(8) respectively (normal range 49-85 ng/3 x 10(8) plt). These results are consistent with a deficiency of platelet PGHS activity. The level of the constitutive platelet PGHS-1 and TXA2 synthase (TXAS) proteins were determined on platelet microsomal fractions by Western blot analysis using affinity-purified polyclonal antibodies highly specific for human PGHS-1 and TXAS, respectively. In two patients the 70 kD PGHS-1 protein was undetectable, whereas it was normal in the third patient. The 60 kD TXAS band was normal in all three patients. These findings indicate that human platelet PGHS-1 deficiency is due to two types of enzyme defects: type 1 defect is manifested by an undetectable PGHS-1 protein in platelets whereas the type 2 defect is manifested by a normal quantity of PGHS-1 protein which has an impaired catalytic activity.

Familial bleeding tendency with partial platelet thromboxane synthetase deficiency: reorientation of cyclic endoperoxide metabolism.

Three family members from three successive generations presented with a moderate bleeding tendency and a functional platelet defect. They had absent aggregation with arachidonic acid (0.6--3 microM), reversible aggregation with ADP (4 microgram) and cyclic endoperoxide analogues, single wave aggregation only with adrenaline (5.4 microgram) and a prolonged template bleeding time (> min). Malondialdehyde formation was reduced after N-ethylmaleimide stimulation (2--6 nmol/10(9) platelets; control values 8--12 nmol) and serum thromboxane B2 values were reduced (33--101 ng/ml; control values 200--700 ng/ml). When the platelets were incubated with [3H]arachidonic acid the final metabolite of the lipoxygenase pathway (HETE) was produced in normal amounts but the production of thromboxane B2 and HHT was decreased whereas prostaglandin F2a, and E2 and probably D2 were increased. Evidence for enhanced production of prostaglandin D2 was also provided by the rise in the patient's platelet cyclic AMP levels following stimulation with arachidonic acid. The patient's washed platelets stimulated the production of 6-keto PGF 1a by aspirin-pretreated cultured bovine endothelial cells. The plasma levels of 6-keto PGF1a (439--703 pg/ml; normal 181 +/- 46 pg/ml) were raised. The decreased production of thromboxane B2, HHT and malondialdehyde and increased formation of prostaglandin F2a, E2, D2 and of 6-keto PGF1a are compatible with a partial platelet thromboxane synthetase deficiency and reorientation of cyclic endoperoxide metabolism. The markedly prolonged bleeding time would result not only from reduced formation of thromboxane A2 but also from increased production of the aggregation inhibiting prostaglandins PGI2 and PGD2.

[Problems in intensive care posed by imbalance in the protease--protease inhibitor system]. / Problèmes de réanimation posés par le déséquilibre du système protéases--inhibiteurs de protéases.

Proteases have a wide range of functions: digestion (pancreatic proteases), protein catabolism (lysosomal proteases), blood coagulation, immune defences (complement), cellular division and proliferation, generation of biologically active oligopeptides (kinins, hormones) from inactive polypeptide precursors, inactivation of these oligopeptides, etc. The body protects itself against its own proteases, either by confining them to a given compartment (lysosome), by synthesising them in the form of inactive precursors (trypsinogen, prothrombin, etc.), or by synthesising proteins with an antiprotease activity. Any disturbance in one of the elements of this protective system may lead to severe pathological consequences: acute hemorrhagic pancreatitis with shock, coagulation disturbances (deficient hepatic synthesis of coagulation factors, congenital antithrombin III deficiency), angioneurotic oedema (congenital deficiency of C'l esterase inhibitor) pulmonary emphysema (local secretion of leukocyte elastase, congenital deficiency of alpha a-antitrypsin).

Assay of Fletcher factor (plasma prekallikrein) using an artificial clotting reagent and a modified chromogenic assay.

An artificial clotting reagent lacking in Fletcher factor (plasma prekallikrein, PPK) was made by mixing human plasma, activated by 5 mg/ml of celite, then kept 16 hours at 37 degrees to destroy most of the plasma kallikrein, plus rabbit plasma (which is devoid of XII and Fletcher activity). Chromogenic assay using a tripeptide substrate was also modified to exclude the interference of the endogenous contact factors. Celite eluate was used instead of kaolin or dextran sulphate for the activation. Using both these methods, it is possible to distinguish between Fletcher trait (PPK deficiency) and other contact factors such as factor XII and HMWK deficiencies, which do not activate with kaolin or dextran sulphate. These simple clotting and enzymatic assays give specific and well correlated results for PPK estimation.

Demonstration of granulocytic proteases in plasma of patients with acute leukemia and septicemia with coagulation defects.

To show whether direct proteolysis of coagulation factors may play a role in patients with so-called consumption coagulopathy, granulocytic neutral proteases in the plasma of patients with acute myelocytic leukemia and septicemia were assayed by one- and two-dimensional Laurell electrophoresis. Complexes between serum alpha1-antitrypsin and elastase-like granulocytic protease could be demonstrated in those patients with acute myelocytic leukemia and septicemia who also had moderate or severe coagulation defects. Despite the presence of a high antiprotease potential, addition of the elastase-like enzyme to normal plasma resulted in coagulation defects in vitro comparable to those seen in the patients. These results and the ability of the elastase-like protease to destroy isolated clotting factors suggested that in certain types of coagulation factor deficiencies direct proteolysis rather than consumption of clotting factors due to disseminated intravascular coagulation may be operational.