Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage.

Agents that restore vascular patency in stroke also increase the risk of intracerebral hemorrhage (ICH). As Factor IXa is a key intermediary in the intrinsic pathway of coagulation, targeted inhibition of Factor IXa-dependent coagulation might inhibit microvascular thrombosis in stroke without impairing extrinsic hemostatic mechanisms that limit ICH. A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site. In a modified cephalin clotting time assay, in vivo administration of Factor IXai caused a dose-dependent increase in time to clot formation (3.6-fold increase at the 300 micrograms/kg dose compared with vehicle-treated control animals, P < 0.05). Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections. At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH. Factor IXai was cerebroprotective even when given after the onset of stroke, indicating that microvascular thrombosis continues to evolve (and may be inhibited) even after primary occlusion of a major cerebrovascular tributary.     

Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors

The regulation of human Factor IXa was studied in vitro in human and mouse plasma and in vivo in the mouse. In human plasma, approximately 60% of the 125I-Factor IXa was bound to antithrombin III (ATIII) by 2 h, with no binding to alpha 2-macroglobulin or alpha 1-proteinase inhibitor, as assessed by gel electrophoresis and IgG- antiproteinase inhibitor-Sepharose beads. In the presence of heparin, virtually 100% of the 125I-Factor IXa was bound to ATIII by 1 min. The distribution of 125I-Factor IXa in mouse plasma was similar. The clearance of 125I-Factor IXa was rapid (50% clearance in 2 min) and biphasic and was inhibited by large molar excesses of ATIII-thrombin and alpha 1-proteinase inhibitor-trypsin, but not alpha 2-macro-globulin-trypsin; it was also inhibited by large molar excesses of diisopropylphosphoryl - (DIP-) Factor Xa, DIP-thrombin, and Factor IX, but not by prothrombin or Factor X. The clearance of Factor IX was also rapid (50% clearance in 2.5 min) and was inhibited by a large molar excess of Factor IX, but not by large molar excesses of Factor X, prothrombin, DIP-Factor Xa, or DIP-thrombin. Electrophoresis and IgG- antiproteinase inhibitor-Sepharose bead studies confirmed that by 2 min after injection into the murine circulation, 60% of the 125I-Factor IXa was bound to ATIII. Organ distribution studies with 125I-Factor IXa demonstrated that most of the radioactivity was in the liver. These studies suggest that Factor IXa binds to at least two classes of binding sites on endothelial cells. One site apparently recognizes both Factors IX and IXa, but not Factor X, Factor Xa, prothrombin, or thrombin. The other site recognizes thrombin, Factor Xa, and Factor IXa, but not the zymogen forms of these clotting factors. After this binding, Factor IXa is bound to ATIII and the complex is cleared from the circulation by hepatocytes.     

A coagulation pathway on bovine aortic segments leading to generation of Factor Xa and thrombin

Previous studies have demonstrated the binding of Factors IX and IXa to cultured bovine aortic endothelial cells. The present study examines the interaction of Factors IX, IXa, and Xa with the luminal surface of calf aortas, shown by microscopic examination to have a continuous layer of endothelium. Radioimmunoassay of Factor IX showed that 74 fmol/10(6) cells of Factor IX could be eluted from freshly prepared aortic segments. Binding of 3H-Factors IX and IXa to aortic segments was saturable, and comparable to binding in previous studies using cultured endothelial cells. Preincubation of aortic segments with 3H-Factor IXa and von Willebrand factor (VWF)/Factor VIII, followed by washing and addition of Factor X, resulted in formation of Factor Xa. The addition of prothrombin to these activation mixtures resulted in formation of thrombin. Exogenous phospholipid and Factor V were not required for Factor X and prothrombin activation on the intact native endothelium. Incubation of 125I-Factor Xa with the vessel segments resulted in most of the tracer being complexed with antithrombin III originally present on the aortic segment (3.8 pmol antithrombin III/10(6) cells). The Factor Xa-antithrombin III complex was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis exclusively in the supernatants. 125I-Factor Xa not complexed with antithrombin III bound specifically to the vessel segment. The time course of binding was biphasic, consisting of an initial more rapid reversible phase followed by a slower irreversible phase. The latter phase correlated with the formation of a covalent complex (Mr, 76,000) between 125I-Factor Xa and a vessel-localized protein presumably distinct from antithrombin III. The activation of prothrombin by vessel-bound Factor Xa was inhibited by anti-bovine Factor V IgG, suggesting that there is interaction of Factor Xa with a Factor V-like molecule provided by the endothelial cell surface. Addition of antibody to antithrombin III prevented formation of Factor Xa-antithrombin III and thrombin-antithrombin III complexes in the supernatant and increased apparent thrombin activity 30-50-fold. These studies demonstrate that freshly obtained vessels with a continuous layer of native endothelium can support activation of Factor X and prothrombin: vessel-bound Factor IXa can activate Factor X in the presence of VWF/Factor VIII. Factor Xa can also bind to the vessel and participate in the activation of prothrombin. The apparent efficiency of prothrombin activation, however, is dampened by the presence of functional antithrombin III on the vessel wall.     

Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis

Formation of fibrin is critical for limiting blood loss at a site of blood vessel injury (hemostasis), but may also contribute to vascular thrombosis. Hereditary deficiency of factor XII (FXII), the protease that triggers the intrinsic pathway of coagulation in vitro, is not associated with spontaneous or excessive injury-related bleeding, indicating FXII is not required for hemostasis. We demonstrate that deficiency or inhibition of FXII protects mice from ischemic brain injury. After transient middle cerebral artery occlusion, the volume of infarcted brain in FXII-deficient and FXII inhibitor-treated mice was substantially less than in wild-type controls, without an increase in infarct-associated hemorrhage. Targeting FXII reduced fibrin formation in ischemic vessels, and reconstitution of FXII-deficient mice with human FXII restored fibrin deposition. Mice deficient in the FXII substrate factor XI were similarly protected from vessel-occluding fibrin formation, suggesting that FXII contributes to pathologic clotting through the intrinsic pathway. These data demonstrate that some processes involved in pathologic thrombus formation are distinct from those required for normal hemostasis. As FXII appears to be instrumental in pathologic fibrin formation but dispensable for hemostasis, FXII inhibition may offer a selective and safe strategy for preventing stroke and other thromboembolic diseases.     

Role of Factor XII in hemostasis and thrombosis: clinical implications

The plasma coagulation system reacts quickly to limit blood loss from injury sites but also contributes to vascular thrombosis. In current models of hemostatic balance, normal coagulation and thrombosis represent two sides of the same coin, however, recent data from gene-deleted murine models have challenged this dogma. Deficiency of coagulation Factor XII (Hageman factor), a serine protease that initiates the intrinsic pathway of coagulation, severely impairs arterial thrombus formation but is not associated with excessive bleeding. These findings suggest that fibrin-generating mechanisms that operate during pathologic thrombus formation involve pathways distinct from those that are active during normal hemostasis. As Factor XII selectively contributes to thrombus formation in occlusive disease, but not to normal hemostasis, inhibition of this protease may offer a novel treatment strategy for prevention of arterial thrombosis with minimal or no risk of bleeding.     

Characterization of the clotting activities of structurally different forms of activated factor IX. Enzymatic properties of normal human factor IXa alpha, factor IXa beta, and activated factor IX Chapel Hill.

Two structurally different forms of activated human Factor IX (Factor IXa alpha and IXa beta) have been previously reported to have essentially identical clotting activity in vitro. Although it has been shown that activated Factor IX Chapel Hill, an abnormal Factor IX isolated from the plasma of a patient with mild hemophilia B, and normal Factor IXa alpha are structurally very similar, the clotting activity of activated Factor IX Chapel Hill is much lower (approximately fivefold) than that of normal Factor IXa beta. In the present study we have prepared activated Factor IX by incubating human Factor IX with calcium and Russell's viper venom covalently bound to agarose. Fractionation of the activated Factor IX by high-performance liquid chromatography demonstrated the presence of both Factors IXa alpha and IXa beta. On the basis of active site concentration, determined by titration with antithrombin III, the clotting activities of activated Factor IX Chapel Hill and IXa alpha were similar, but both activities were less than 20% of the clotting activity of Factor IXa beta. Activated Factor IX activity was also measured in the absence of calcium, phospholipid, and Factor VIII, by determination of the rate of Factor X activation in the presence of polylysine. In the presence of polylysine, the rates of Factor X activation by activated Factor IX Chapel Hill, Factor IXa alpha, and Factor IXa beta were essentially identical. We conclude that the clotting activity of activated Factor IX Chapel Hill is reduced when compared with that of Factor IXa beta but essentially normal when compared with that of Factor IXa alpha.     

Effects of factor IX or factor XI deficiency on ferric chloride-induced carotid artery occlusion in mice.

Factor XI (FXI) and factor IX (FIX) are zymogens of plasma serine proteases required for normal hemostasis. The purpose of this work was to evaluate FXI and FIX as potential therapeutic targets by means of a refined ferric chloride (FeCl(3))-induced arterial injury model in factor-deficient mice. Various concentrations of FeCl(3) were used to establish the arterial thrombosis model in C57BL/6 mice. Carotid artery blood flow was completely blocked within 10 min in C57BL/6 mice by application of 3.5% FeCl(3). In contrast, FXI- and FIX-deficient mice were fully protected from occlusion induced by 5% FeCl(3), and were partially protected against the effect of 7.5% FeCl(3). The protective effect was comparable to very high doses of heparin (1000 units kg(-1)) and substantially more effective than aspirin. While FXI and FIX deficiencies were indistinguishable in the carotid artery injury model, there was a marked difference in a tail-bleeding-time assay. FXI-deficient and wild-type mice have similar bleeding times, while FIX deficiency was associated with severely prolonged bleeding times (>5.8-fold increase, P < 0.01). Given the relatively mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable strategy for treating or preventing thrombus formation.     

Prevention of catheter thrombosis increases survival, but does not modify lung injury in rats receiving long-term parenteral nutrition

It has previously been shown that rats receiving total parenteral nutrition (TPN) or each component of TPN die within 40 days of treatment. Central catheter thrombosis and lung injury were constant findings. The aim of the present study was to examine the impact of central thrombosis on lung injury and survival in rats receiving long-term parenteral nutrition. In the first part of the study TPN was infused via the jugular vein and incidence of central venous thrombosis and rate of survival were recorded. Addition of low molecular weight heparin (LMWH) reduced central thrombosis from 6 out of 7 animals to 2 out of 7 animals (p=0.027) and increased survival from 17.1+/-4.5 days to 32.4+/-4.9 days (p=0.04). In the second part of the study four infusion groups were established. Group 1 (controls) received saline 100 mL/kg/day via the jugular vein (n=6). Group 2 received Intralipid 40 mL/kg/day via the jugular vein (n = 7). Group 3 received Intralipid 40 mL/kg/day via the portal vein (n = 7). Group 4 received Intralipid 40 mL/kg/day with added LMWH 70 U/kg/day (n = 7). Lung injury and occurrence of central thrombosis were investigated. Lung injury was assessed by measuring pulmonary arterial pressure (Ppa), clearance of serotonin by the vascular endothelium and the capillary filtration coefficient (CFC). Either infusion via the portal vein or the addition of LMWH to the infusion via the jugular vein prevented central thrombus formation, but the lung injury was not modified by this method compared with infusing Intralipid via the jugular vein without LMWH. In conclusion, central thrombus formation contributes to death in rats receiving parenteral nutrition. The mechanism of the injurious effect of central thrombosis remains unknown, but central thrombus formation seems not to increase lung injury caused by Intralipid.     

Prevention of catheter thrombosis increases survival,but does not modify lung injury in rats receiving long-term parenteral nutrition

It has previously been shown that rats receiving total parenteral nutrition (TPN) or each component of TPN die within 40 days of treatment. Central catheter thrombosis and lung injury were constant findings. The aim of the present study was to examine the impact of central thrombosis on lung injury and survival in rats receiving long-term parenteral nutrition. In the first part of the study TPN was infused via the jugular vein and incidence of central venous thrombosis and rate of survival were recorded. Addition of low molecular weight heparin (LMWH) reduced central thrombosis from 6 out of 7 animals to 2 out of 7 animals (p=0.027) and increased survival from 17.1±4.5 days to 32.4±4.9 days (p=0.04). In the second part of the study four infusion groups were established. Group 1 (controls) received saline 100?mL/kg/day via the jugular vein (n=6). Group 2 received Intralipid&;lt;formula&;gt;^®&;lt;/formula&;gt; 40?mL/kg/day via the jugular vein (n=7). Group 3 received Intralipid 40?mL/kg/day via the portal vein (n=7). Group 4 received Intralipid 40?mL/kg/day with added LMWH 70?U/kg/day (n=7). Lung injury and occurrence of central thrombosis were investigated. Lung injury was assessed by measuring pulmonary arterial pressure (Ppa), clearance of serotonin by the vascular endothelium and the capillary filtration coefficient (CFC). Either infusion via the portal vein or the addition of LMWH to the infusion via the jugular vein prevented central thrombus formation, but the lung injury was not modified by this method compared with infusing Intralipid via the jugular vein without LMWH. In conclusion, central thrombus formation contributes to death in rats receiving parenteral nutrition. The mechanism of the injurious effect of central thrombosis remains unknown, but central thrombus formation seems not to increase lung injury caused by Intralipid.     

Endothelial cells and vascular hemostasis]

Procoagulant, anticoagulant, and fibrinolytic activities are associated with endothelial cells and involve the production, secretion, and receptor mediated binding of proteins involved in these processes. The procoagulant aspect of endothelial cells function involves the production and release of von Willebrand Factor(vWF), the production of tissue factor, and the presence of Factor IX/IXa receptors on the cell surface. Secretion of vWf will promote the initial steps in thrombus formation by supporting platelet-platelet interaction and platelet-subendothelial matrix adhesion. Tissue factor which is undetectable in resting cells appears after exposure to various cytokines and initiates factor VIIa activation of factors IX and X. Receptors of Factor IX/IXa are also present and mediate the assembly of the prothrombinase complex on the endothelial cell surface. The anticoagulant pathway involves the cell surface protein thrombomodulin, protein C and its cofactor protein S. Thrombomodulin binds thrombin which activates protein C which in the presence of protein S cleaves and inactivates Factors V and VIII. Inactivation of these two coagulation cofactors halts the coagulation. Finally, endothelial cells also play a pivotal role in the fibrinolytic system. Production and regulated secretion of tissue plasminogen activator creates a profibrinolytic state in the endothelial cell environment. In addition, receptors for plasminogen and urokinase are also present, constituting a cell surface mediated fibrinolytic pathway. Plasminogen activator inhibitor type I, the primary inhibitor of tPA, is also produced by endothelial cells. Thus endothelial cells can promote and inhibit fibrinolysis, depending on the prevailing environmental conditions.     

Effect of thromboxane receptor antagonists on venous thrombosis in rats

The activities of two structurally unrelated thromboxane/prostaglandin endoperoxide receptor antagonists, SQ 30,741 and BM 13,505, were compared to heparin in a model of venous thrombosis. A combination of blood stasis with osmotic and pressure stress was used to induce thrombus formation in the vena cava of anesthetized rats. Intravenous infusions of SQ 30,741 (500 micrograms/kg/min) and BM 13,505 (50 micrograms/kg/min) produced significant (P less than .01) and equivalent reductions in thrombus mass of 58 and 56%, respectively. Thrombus reduction in response to heparin (50 U/kg) was greater (95%; P less than .001) than in response to the thromboxane antagonists. Either lower doses of SQ 30,741 (50 and 100 micrograms/kg/min) or aspirin (30 and 60 mg/kg) were ineffective in altering thrombus formation. However, a subthreshold dose of SQ 30,741 (100 micrograms/kg/min) increased (P less than .01) the antithrombotic activity obtained with both threshold (0.5 U/kg) and subthreshold (0.3 U/kg) doses of heparin. SQ 30,741 (500 micrograms/kg/min) did not change activated partial thromboplastin times or inhibit platelet loss induced by contact activation in response to kaolin in vivo. This suggests that SQ 30,741 does not interfere with components of the coagulation cascade that are not dependent on platelet factors. The extent of thromboxane antagonism achieved with SQ 30,741 (50 and 500 micrograms/kg/min) and BM 13,505 (50 micrograms/kg/min) was determined from parallel shifts in dose-dependent U-46,619-induced vasoconstriction in vivo (approximately 200- and 1300-fold, respectively, for SQ 30,741 and 200-fold for BM 13,505). These data demonstrate that thromboxane antagonists inhibit venous thrombosis partially, but only at doses producing near complete receptor inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Normal saline versus heparin flush for maintaining central venous catheter patency during apheresis collection of peripheral blood stem cells (PBSC)

Thrombotic occlusion is frequently a complication of central venous catheters (CVCs). The original designers and producers of CVCs recommended heparin flush regimens to prevent thrombosis and maintain patency. This has become standard practice although no studies have demonstrated a relationship between heparin flushing and reduction of catheter thrombosis. Many consider the routine use of heparin flushing innocuous. However, serious complications including drug interactions and heparin induced thrombocytopenia and thrombosis syndrome (HITS) have been reported in association with heparin flushing. Numerous studies comparing heparin to saline flushing in peripheral devices suggest equal rates of thrombotic occlusions. The purpose of this study was to examine the incidence of thrombotic occlusions in CVCs using heparin compared to saline flushing. The study involved 78 cancer patients undergoing apheresis collection for peripheral blood stem cells; 29 received saline flushes and 49 received heparin (100 U/ml of saline) flushes. Study endpoints included slow apheresis flow rate (< 50 ml/min), urokinase use for thrombolysis, and radiographic evidence of catheter thrombosis. No significant differences were found for any endpoint between the two groups. These findings suggest saline may be as effective as heparin for maintaining patency of CVCs.     

Repeated doses of oral and subcutaneous heparins have similar antithrombotic effects in a rat carotid arterial model of thrombosis

Although heparins are usually injected intravenously or subcutaneously, antithrombotic activity is observed in rat models following single oral heparin doses. Since repetitive dosing is usually needed for thromboprophylaxis, study objectives were to determine whether repetitive oral heparin prevented arterial thrombosis and to compare effectiveness to subcutaneous administration. Wistar rats were given subcutaneous or oral unfractionated heparin ([UFH] 1 mg/kg per 48 h), low-molecular-weight heparin ([LMWH] tinzaparin, 0.1 mg/kg per 12 h), or saline for 30 days. On the last day, thrombosis was initiated by placing 30% FeCl(3)-soaked filter paper on the distal carotid. Subsequent flow measurements, for a 60-minute period, included recorded time of initial thrombus formation (time till thrombus begins [TTB]), and time until carotid occlusion (time till occlusion [TTO]). The formed thrombus was dried and weighed. The activated partial thromboplastin time (aPTT), anti-factor Xa, and antithrombin activity were determined from the plasma. Both oral and subcutaneous heparins significantly increased TTB and TTO. Time of initial thrombus formations were 12.6 ± 1.1, 21.2 ± 2.2, 25.3 ± 3.9, 21.7 ± 3.1, and 21.3 ± 1.7 minutes and TTOs were 29.3 ± 3.6, 54.8 ± 4.0, 60.0 ± 0.3, 56.7 ± 3.3, and 58.3 ± 1.7 minutes (mean ± SEM) for control, subcutaneous UFH, oral UFH, subcutaneous LMWH, and oral LMWH, respectively. Thrombus weight was 2.52 ± 0.29 g in control and was reduced to 43%, 23%, 33%, and 28% of control weight for subcutaneous UFH, oral UFH, subcutaneous LMWH, and oral LMWH, respectively. Thrombus weight was significantly less for oral compared to subcutaneous UFH. The aPTT for oral UFH, and anti-factor Xa activity in the LMWH-treated groups were significantly greater than control (two-tailed t tests). These findings confirm that orally administered heparins are absorbed. Repeated treatment with oral heparin showed similar antithrombotic activity compared to subcutaneous heparin. Oral heparin use for arterial thromboprophylaxis should be further investigated.     

Kinetics of the Factor XIa catalyzed activation of human blood coagulation Factor IX

The kinetics of activation of human Factor IX by human Factor XIa was studied by measuring the release of a trichloroacetic acid-soluble tritium-labeled activation peptide from Factor IX by a modification of a method described for bovine Factor IX activation by Zur and Nemerson (Zur, M., and Y. Nemerson, 1980, J. Biol. Chem., 255:5703-5707). Initial rates of trichloroacetic acid-soluble 3H-release were linear over 10-30 min of incubation of Factor IX (88 nM) with CaCl2 (5 mM) and with pure (greater than 98%) Factor XIa (0.06-1.3 nM), which was prepared by incubating human Factor XI with bovine Factor XIIa. Release of 3H preceded the appearance of Factor IXa activity, and the percentage of 3H released remained constant when the mole fraction of 3H-labeled and unlabeled Factor IX was varied and the total Factor IX concentration remained constant. A linear correlation (r greater than 0.98, P less than 0.001) was observed between initial rates of 3H-release and the concentration of Factor XIa, measured by chromogenic assay and by radioimmunoassay and added at a Factor IX:Factor XIa molar ratio of 70-5,600. Kinetic parameters, determined by Lineweaver-Burk analysis, include Km (0.49 microM) of about five- to sixfold higher than the plasma Factor IX concentration, which could therefore regulate the reaction. The catalytic constant (kcat) (7.7/s) is approximately 20-50 times higher than that reported by Zur and Nemerson (Zur, M., and Y. Nemerson, 1980, J. Biol. Chem., 255:5703-5707) for Factor IX activation by Factor VIIa plus tissue factor. Therefore, depending on the relative amounts of Factor XIa and Factor VIIa generated in vivo and other factors which may influence reaction rates, these kinetic parameters provide part of the information required for assessing the relative contributions of the intrinsic and extrinsic pathways to Factor IX activation, and suggest that the Factor XIa catalyzed reaction is physiologically significant.     

Local thrombin synthesis and fibrin formation in an in vitro thrombosis model result in platelet recruitment and thrombus stabilization on collagen in heparinized blood

The role of the local synthesis of thrombin in platelet recruitment and thrombus stabilization in heparinized blood was examined in vitro. Mural thrombosis was visualized and measured in a thin, rectangular, collagen-coated capillary under controlled rheological conditions by using fluorescence digital videomicroscopy and fluorescence microphotometry. Thrombin activity was inhibited in heparinized blood by the synthetic competitive inhibitor, D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (FPRCH2Cl), resulting in a marked reduction in the rate of platelet accumulation on collagen surfaces, indicating a role for thrombin in platelet recruitment. Similar although lesser effects were observed by reducing thrombin synthesis with antibodies to factors II and X. To decouple the role of thrombin in platelet recruitment by direct stimulation of platelet activity from its role in thrombus stabilization via fibrin formation, thrombosis was measured in heparinized blood treated with the tetrapeptide glycyl-prolyl-arginyl-proline, which inhibits fibrin monomer assembly into fibrin. The ultimate level but not the initial rate of platelet accumulation was reduced markedly, indicating a role for fibrin in thrombus stabilization against hemodynamic forces. Scanning electron micrographs demonstrated fibrin stands in the heparinized control samples but not in the heparinized samples with glycyl-prolyl-arginyl-proline. These results demonstrate a role for the local action of thrombin synthesized on the surfaces of thrombi even under conditions when the thrombin exerts no bulk effect, such as under heparin anticoagulation. Furthermore, this role appears to be a result of both platelet recruitment and thrombus stabilization.     

Potential role of recombinant factor VIIa as a hemostatic agent

Recombinant factor VIIa (rFVIIa) has been shown to induce hemostasis in hemophilia patients with inhibitors against factor VIII or factor IX independent of factor VIII/factor IX. Factor VIIa binds to tissue factor (TF) exposed at the site of injury and generates, through factor X activation on the TF-bearing cells, enough thrombin to activate factors VIII, V, and XI, as well as platelets. The thrombin-activated platelets provided a perfect template for binding of activated factors VIII, IX, and V, further activation of factor X, and thrombin generation. Factor VIIa in high concentrations binds to thrombin-activated platelets and is capable of activating factor X, thereby generating thrombin independent of the presence of factor VIII or factor IX. Accordingly, rFVIIa has been shown to initiate hemostasis in severe hemophilila patients with inhibitors subjected to major surgery and suffering from serious limb- and life-threatening bleeding. Since rFVIIa enhances thrombin generation-thereby providing the formation of tight, stable fibrin hemostatic plugs resistance to premature lysis-it should be hemostatic in other situations characterized by impaired thrombin generation. A hemostatic effect has been reported in patients with various platelet disorders and factor XI deficiency. Further, a hemostatic effect of rFVIIa has been reported in patients subjected to trauma and extensive surgery who have developed profuse, excessive bleeding resulting in hemodilution and changes in coagulation patterns. rFVIIa was developed to treat bleeding in hemophilia patients with inhibitors against factor VIII or factor IX and has been shown to induce effective hemostasis in most such patients and also in life- and limb-threatening bleeding. It has also been used successfully to stop bleeding in patients who do not have hemophilia but who do have acquired antibodies against factor VIII (acquired hemophilia). rFVIIa initiates hemostasis by forming a complex with TF exposed as a result of vessel wall injury. Pharmacologic doses of rFVIIa can enhance thrombin generation on platelets that are already thrombin-activated, resulting in the formation of full thrombin burst. By enhancing thrombin generation, rFVIIa helps to form tight, stable, fibrin plugs resistant to premature fibrinolysis. This also maintains hemostasis in the absence of factor VIII or factor IX. Pharmacologic doses of rFVIIa may accordingly be of benefit in producing hemostasis in situations other than hemophilia characterized by profuse bleeding and impaired thrombin generation. There is now clinical experience indicating a hemostatic effect in patients with thrombocytopenia and functional platelet defects. rFVIIa has also been successfully used in acute trauma patients with profuse bleedings and in other bleeding situations.     

In vivo thrombus formation

Summary.  Thrombus formation, including platelet adhesion, activation, secretion and aggregation as well as tissue factor-initiated thrombin generation and fibrin formation, has been studied in the past using in vitro systems, often with isolated components. Given the complexity of hemostasis and thrombosis, many of the concepts that have been developed to explain these processes are being revisited by studying thrombus formation in live animals using intravital microscopy and genetically altered mice. Although much of the dogma that has evolved has been confirmed by in vivo studies of thrombus formation, there have also been conflicts between old concepts and new direct observations. In vivo studies of the initiation of thrombus formation, platelet accumulation and thrombin generation have provided evidence for the participation of novel proteins and identified new pathways and mechanisms.     

Potentiation of the antithrombotic effect of prostacyclin by simultaneous administration of aminophylline in a canine model of coronary artery thrombosis

The antithrombotic efficacy of prostacyclin (PGI2) when administered in conjunction with the phosphodiesterase inhibitor aminophylline was evaluated in a canine model in which coronary artery thrombosis was induced by electrical stimulation of the intimal surface of the left circumflex (LCX) coronary artery. Infusions of PGI2 (25 or 50 ng/kg/min) into the left atrial appendage and aminophylline (20 micrograms/kg/min) or ethylene diamine into the left jugular vein were initiated 10 min before the start of LCX coronary artery stimulation and continued for the 6-hr stimulation period. Every animal in the control (Tris buffer plus ethylene diamine, n = 7), PGI2 (25 ng/kg/min) only (n = 6) and aminophylline only (n = 7) groups developed completely occlusive coronary artery thrombi. In contrast, none of the animals receiving PGI2 (25 ng/kg/min) plus aminophylline or PGI2 (50 ng/kg/min) plus aminophylline underwent occlusive thrombus formation. The average thrombus mass developed in response to intimal injury of the LCX coronary artery was 57 +/- 14 mg (X +/- S.E.M.) in the control group. Aminophylline administration in conjunction with PGI2 infusion at doses of 25 and 50 ng/kg/min significantly reduced thrombus mass to 11 +/- 2 and 10 +/- 1 mg, respectively (P less than .05). PGI2 (25 ng/kg/min) plus aminophylline reduced mean arterial pressure by 12% from 116 +/- 5 to 102 +/- 4 mm Hg. These data demonstrate that the combined administration of aminophylline with low-dose PGI2 provides antithrombotic efficacy while minimizing the detrimental hemodynamic effects of large-dose PGI2 administration.     

Protease nexin-2/amyloid beta protein precursor. A tight-binding inhibitor of coagulation factor IXa.

Protease nexin-2/amyloid beta protein precursor (PN-2/A beta PP) is an abundant, secreted platelet protein which is a potent inhibitor of coagulation Factor XIa. We examined other potential anticoagulant activities of PN-2/A beta PP. Purified Kunitz protease inhibitor domain of PN-2/A beta PP and PN-2/A beta PP itself were found to prolong the coagulation time of plasma and pure Factor IXa. The Kunitz protease inhibitor domain also inhibited the ability of Factor IXa to activate Factor X. PN-2/A beta PP inhibited Factor IXa with a Ki of 7.9 to 3.9 x 10(-11) M in the absence and presence of heparin, respectively. When the second-order rate constant of PN-2/A beta PP's inhibition of Factor IXa (2.7 x 10(8) M-1min-1) was compared to that of antithrombin III (3.8 x 10(6) M-1min-1), PN-2/A beta PP was at least a 71-fold more potent inhibitor of Factor IXa than antithrombin III. PN-2/A beta PP formed a complex with Factor IXa as detected by gel filtration and ELISA. The finding that PN-2/A beta PP is a potent inhibitor of Factor IXa could help to explain the spontaneous intracerebral hemorrhages seen in patients with hereditary cerebral hemorrhage with amyloidosis Dutch-type where there is an extensive accumulation of PN-2/A beta PP in their cerebral blood vessels.     

Comparison of Heparin and Saline for Prevention of Central Venous Catheter Occlusion in Pediatric Oncology: A Systematic Review and Meta-Analysis

ObjectiveThe management of central venous catheter (CVC) occlusion remains an area without clear evidence-based guidelines. Studies have been conducted that compare the use of heparin and normal saline for reducing thrombosis, but the evidence is not strong enough to suggest a significant advantage of one over the other. Therefore, the study aimed to assess the effectiveness of heparin and normal saline flushing in preventing CVC occlusion in pediatric patients with cancer.Data SourcesA comprehensive search was conducted in PubMed, Web of Science, Cochrane, MEDLINE, CINAHL, Embase, World Health Organization International Clinical Trials Registry Platform, and ClinicalTrials.gov platform using specific keywords. The search was conducted until March 2022. Five randomized controlled trials are included in this study.ConclusionFive studies with a total of 316 pediatric cancer patients met the inclusion criteria. The studies were found to be heterogeneous due to variations in the types of cancer, heparin concentration, flushing frequency of CVCs, and methods used to measure occlusion. Despite these differences, there was no significant difference in the effect of flushing with heparin and normal saline in preventing CVC occlusion. The analysis revealed that normal saline is as effective as heparin in preventing CVC occlusion among pediatric cancer patients.Implications for Nursing PracticeThis systematic review and meta-analysis demonstrated that there is no significant difference between the use of heparin and normal saline flushing in preventing CVC occlusion among pediatric cancer patients. Considering the potential risks of heparin, the use of normal saline flushing may be recommended to prevent CVC obstruction.