Liver-specific γ-glutamyl carboxylase-deficient mice display bleeding diathesis and short life span.

Vitamin K is a fat-soluble vitamin that plays important roles in blood coagulation and bone metabolism. One of its functions is as a co-factor for γ-glutamyl carboxylase (Ggcx). Conventional knockout of Ggcx causes death shortly after birth in homozygous mice. We created Ggcx-floxed mice by inserting loxP sequences at the sites flanking exon 6 of Ggcx. By mating these mice with albumin-Cre mice, we generated Ggcx-deficient mice specifically in hepatocytes (Ggcx(Δliver/Δliver) mice). In contrast to conventional Ggcx knockout mice, Ggcx(Δliver/Δliver) mice had very low activity of Ggcx in the liver and survived several weeks after birth. Furthermore, compared with heterozygous mice (Ggcx(+/Δliver) ), Ggcx(Δliver/Δliver) mice had shorter life spans. Ggcx(Δliver/Δliver) mice displayed bleeding diathesis, which was accompanied by decreased activity of coagulation factors II and IX. Ggcx-floxed mice can prove useful in examining Ggcx functions in vivo.

Association between tPA therapy and raised early matrix metalloproteinase-9 in acute stroke.

BACKGROUND: Matrix metalloproteinase-9 (MMP9) is expressed in acute ischemic stroke and up-regulated by tissue plasminogen activator (tPA) in animal models. The authors investigated plasma MMP9 and its endogenous inhibitor, tissue inhibitor of metalloproteinase (TIMP1), in tPA-treated and -untreated stroke patients. METHODS: Nonstroke control subjects and consecutive ischemic stroke patients presenting within 8 hours of onset were enrolled. Blood was sampled within 8 hours and at 24 hours, 2 to 5 days and 4 to 6 weeks. MMP9 and TIMP1 were analyzed by ELISA and gel zymography. RESULTS: Fifty-two cases (26 tPA treated, 26 tPA untreated) and 27 nonstroke control subjects were enrolled. Hyperacute MMP9 was elevated in tPA-treated vs tPA-untreated patients (medians 43 vs 28 ng/mL; p = 0.01). tPA therapy independently predicted hyperacute MMP9 after adjustment for stroke severity, volume, and hemorrhagic transformation (p = 0.01). There was a trend toward lower hyperacute TIMP1 levels in tPA-treated vs tPA-untreated patients (p = 0.06). Hyperacute MMP9 was correlated to poor 3-month modified Rankin Scale outcome (r = 0.58, p = 0.0005). CONCLUSION: Tissue plasminogen activator independently predicted plasma matrix metalloproteinase-9 (MMP9) in the first 8 hours after human ischemic stroke. As MMP9 may be an important mediator of hemorrhagic transformation, alternative thrombolytic agents or therapeutic MMP9 inhibition may increase the safety profile of acute stroke thrombolysis.

Von Willebrand factor-cleaving protease and Upshaw-Schulman syndrome.

Vascular endothelial cell (EC)-produced plasma von Willebrand factor (vWF) plays a critical role in primary hemostasis through its action of anchoring platelets onto the injured denuded subendothelial matrices under high shear stress. Unusually large vWF multimers (UL-vWFMs), present in plasma immediately after release from ECs, are most biologically active, but they are soon cleaved and degraded into smaller vWFMs by a specific plasma protease, termed vWF-cleaving protease (vWF-CPase), in normal circulation. Recent studies on the relationship between UL-vWFMs and vWF-CPase, together with its autoantibody (inhibitor) have brought about a clear discrimination between thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Furthermore, a congenital deficiency of this enzyme activity has been shown to cause Upshaw-Schulman syndrome, a complex constitutional bleeding diathesis. Successful purification of vWF-CPase revealed that this enzyme is composed of a single polypeptide with a molecular mass of approximately 190 kd, and its complementary DNA cloning unambiguously indicated that it is uniquely produced in the liver and its gene is located on chromosome 9q34. The messenger RNA of vWF-CPase had a span of 4.6 kb, and its enzyme was designated ADAMTS 13. The predicted complete amino acid sequence of this enzyme consisted of 1427 residues, including a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin-1 repeat (TSP1), a cysteine-rich domain, an ADAMTS spacer, 7 additional TSP1 repeats, and 2 CUB domains.

Upshaw-Schulman syndrome revisited: a concept of congenital thrombotic thrombocytopenic purpura.

Upshaw-Schulman syndrome (USS) is a congenital bleeding disorder characterized by repeated episodes of thrombocytopenia and microangiopathic hemolytic anemia that respond to infusions of fresh frozen plasma. Inheritance of USS has been thought to be autosomal recessive, because 2 siblings in the same family are often affected but their parents are asymptomatic. Recently, chronic relapsing thrombotic thrombocytopenic purpura (CR-TTP), reported almost exclusively in adults, was shown to be caused by inherited or acquired deficiency in the activity of a plasma von Willebrand factor-cleaving protease (vWF-CPase). The pathogenesis of USS is unknown, and a relationship between CR-YEP and USS has not been reported. We studied 3 unrelated USS patients (ST, SY, and KI) who presented with severe indirect neonatal hyperbilirubinemia. All 3 patients had undetectable vWF-CPase activity, and the inhibitors to vWF-CPase were all negative. In their parents with no clinical symptoms, vWF-CPase activities as a percentage of control samples (mother/father) were 17/20 for ST, 60/45 for SY, and 36/5.6 for KI. Thus, USS and vWF-CPase activity appear to be coinherited as autosomal recessive traits. Transfusion of fresh frozen plasma in 2 patients (ST and SY) resulted in the expected maximal increment of approximately 7% to 8% in vWF-CPase activity at 1 to 4 hours, but the levels became less than 3% within 2 days. After this decrease, platelet counts increased, plateaued in the normal range at 10 to 12 days, and declined thereafter. Thus, the 2 to 3 weeks of therapeutic benefit from plasma infusions will be discussed in relation to the intravascular lifetime of vWF-CPase.

Deficient activity of von Willebrand factor-cleaving protease in patients with Upshaw-Schulman syndrome.

We identified unusually large von Willebrand factor (vWF) multimers caused by deficient activity of vWF-cleaving protease in 2 patients with Upshaw-Schulman syndrome. The autoantibodies that inhibited the protease activity were not detected in the plasma of either patient. Periodic fresh-frozen plasma transfusion was effective for management of the hemolysis and thrombocytopenia. We detected enriched enzyme activity in a particular plasma fraction, although molecular cloning of this specific protease is needed to determine a more detailed pathogenesis and to develop new therapeutic approaches.

Human platelet signaling defect characterized by impaired production of inositol-1,4,5-triphosphate and phosphatidic acid and diminished Pleckstrin phosphorylation: evidence for defective phospholipase C activation.

Signal transduction on platelet activation involves phosphoinositide-specific phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositides and formation of inositol-1,4,5-triphosphate [I(1,4,5)P3], which mediates Ca2+ mobilization, and diacylglycerol (DG), which activates protein kinase C (PKC) to phosphorylate a 47-kD protein (Pleckstrin). We studied these events in two related patients previously reported (Blood 74:664, 1989) to have abnormal aggregation and 14C-serotonin secretion, and impaired intracellular Ca2+ mobilization in response to several agonists. Thrombin-induced I(1,4,5)P3 and phosphatidic acid formation were diminished. Pleckstrin phosphorylation was impaired on activation with thrombin, platelet-activating factor, and ionophore A23187, but was normal with PKC activator 1,2-dioctonyl-sn-glycerol (DiC8). Ca2+ mobilization induced by guanosine triphosphate (GTP) analog guanosine 5'-0-(3 thiotriphosphate) (GTP gamma S) was diminished. Pretreatment with either A23187 or DiC8 did not correct the impaired adenine diphosphate-induced secretion; however, upon stimulation with A23187 plus DiC8, pleckstrin phosphorylation and secretion were normal, indicating that both PKC activation and Ca2+ mobilization are essential for normal secretion. We conclude that these patients have a unique inherited platelet defect in formation of two key intracellular mediators [I(1,4,5)P3 and DG] and in the responses mediated by them due to a defect in postreceptor mechanisms of PLC activation.

Vascular thromboxane formation in hemostasis mechanism: correlation between bleeding time and vascular TXB2 in a patient with congenital platelet cyclo-oxygenase deficiency.

We encountered a patient with congenital platelet cyclo-oxygenase deficiency with normal ability to synthesize vascular prostaglandin I2 (PGI2) and thromboxane A2 (TXA2). The patient's peripheral blood monocytes did not show cyclo-oxygenase (COX) activity, but cultured bone marrow fibroblasts showed COX activity. To determine the mechanism of primary hemostasis in this patient, we examined the effect of oral administration of aspirin (1 g) on bleeding time and thromboxane B2 (TXB2), 6-keto prostaglandin F1 alpha (6-keto-PGF1 alpha) production in the blood emerging from the incision in this patient. The bleeding time was markedly prolonged by the administration of aspirin, and this prolongation was associated with the inhibition of TXB2 in the effluent blood, which seemed to be derived from the vessel wall. These findings suggest that vascular TXA2 production plays an important role in the maintenance of hemostasis.

Mutation of antitrypsin to antithrombin. alpha 1-antitrypsin Pittsburgh (358 Met leads to Arg), a fatal bleeding disorder.

Our previous studies predicted a functional relationship between the plasma proteins alpha 1-antitrypsin and antithrombin III. To elucidate this relationship we investigated the plasma of a 14-year-old boy who had died from an episodic bleeding disorder. A variant alpha 1-antitrypsin was identified in which the methionine at position 358 had been replaced by an arginine. This had converted the alpha 1-antitrypsin from its normal function as an inhibitor of elastase to that of an inhibitor of thrombin. This finding indicates that the reactive center of alpha 1-antitrypsin is methionine 358, which acts as a bait for elastase, just as the normal reactive center of antithrombin III is arginine 393, which acts as a bait for thrombin. The independence of the new thrombin inhibitor from heparin control explains the bleeding disorder; it also indicates that heparin normally acts directly on antithrombin III, revealing its inherent inhibitory activity. The episodic nature of the bleeding was a consequence of the mutant protein's being an acute-phase reactant, the level of which increased several-fold after trauma.

Biochemical studies in Hermansky-Pudlak syndrome.

In order to find the basic defect in the Hermansky-Pudlak Syndrome (HPS), biochemical studies of platelets and leucocytes were undertaken. Glutathione levels of platelets were normal and regeneration of GSH similar to controls occurred after incubation with diamide (a specific agent for GSH oxidation). Phospholipid and fatty acid composition of HPS platelets was normal. The amount of peroxides found in platelet membranes was not elevated. A subnormal aggregation with arachidonic acid could be obtained in PRP using a high concentration of arachidonic acid (2 mM), but normal malondialdehyde levels were measured, suggesting a normal prostaglandin synthesis in HPS platelets. Glutathion peroxidase and p-phenylenediamide-mediated peroxidase (PPD-peroxidase) were normal in leucocytes of 1 HPS patient. Lysosomal enzymes as far as investigated were normal.