Influence of cardiopulmonary bypass on the interaction of recombinant factor VIIa with activated platelets.

Recombinant factor VIIa (rFVIIa) interacts preferentially with coated platelets characterized by a high exposure of phosphatidyl serine (PS), FV, FVIII, FIX, and FX binding, and fibrinogen. Cardiopulmonary bypass (CPB) is known to impair platelet function. In this study, the influence of CPB on formation of coated platelets and the interaction of rFVIIa with the platelets were studied. Blood was either exposed to a closed CPB circuit or obtained from patients undergoing CPB-assisted cardiac surgery, and platelets were analyzed by flow cytometry with and without dual agonist stimulation with thrombin and a GPVI collagen receptor agonist known to induce coated platelet formation. Platelets circulated within a closed CPB circuit did not spontaneously form coated platelets. Dual agonists stimulation caused formation of coated platelets at a reduced level compared to pre-CPB level (51 +/- 21% vs. 80 +/- 17% before CPB, p < .001). The rFVIIa interaction with the coated platelets was not impaired after CPB. Platelets isolated from patients undergoing CPB-assisted cardiac surgery also formed coated platelets only after dual agonist stimulation but to the same level as before surgery (76 +/- 8% vs. 83 +/- 14% before surgery, p = .17, n = 10). rFVIIa interaction with the coated platelets was not impaired after surgery. No spontaneous rFVIIa-binding platelets were found. The data indicate that CPB exposure in vivo does not compromise the platelet-dependent effects of rFVIIa either by spontaneous formation of coated platelets, thereby limiting the risk of systemic coagulation, or by impairing rFVIIa interaction with the agonist-induced coated platelets, thereby retaining the hemostatic potential of rFVIIa after CPB.

Angiotensin converting enzyme-regulated, noncholinergic sympathoadrenal catecholamine release mediates the cardiovascular actions of human 'new pressor protein' related to coagulation beta-factor XIIa.

BACKGROUND: Human 'new pressor protein' (NPP), related to coagulation beta-factor XIIa (beta-FXIIa), potently releases sympathoadrenal catecholamines in bioassay rats, with concurrent elevation of systolic and diastolic blood pressure (SBP/DBP) and heart rate (HR). Elevated plasma NPP/beta-FXIIa levels in hypertensive anephric pediatric patients on hemodialysis associated with fluid status and blood pressure changes were previously reported, suggesting that NPP/beta-FXIIa contributed to their hypertension. OBJECTIVE: To investigate the mechanism of action of NPP/beta-FXIIa. METHODS: Hemodynamic and sympathoadrenal responses to NPP (20 microL plasma equivalent/rat) or coagulation beta-FXIIa (300 ng/kg intravenously) were measured in rats treated with pentolinium (ganglion blockade [+GB]) and/or captopril (+CAP; angiotensin converting enzyme [ACE] inhibition). RESULTS: In controls not receiving GB or CAP (-GB-CAP), NPP/beta-FXIIa raised plasma epinephrine (E) sixfold, SBP/DBP by 14/8 mmHg and HR by 15 beats/min. With blockade of the cholinergic pathway to the sympathoadrenal system (+GB), basal E, norepinephrine (NE), SBP, DBP and HR all dropped. However NPP/beta-FXIIa remained capable of raising E 20-fold, NE fourfold, SBP/DBP by 27/11 mmHg and HR by 20 beats/min, suggesting that it acted through a 'noncholinergic' mechanism. With +CAP alone, NPP/beta-FXIIa raised plasma E 18-fold, NE threefold, SBP/ DBP by 29/8 mmHg and HR by 73 beats/min, implicating an ACE-regulated 'peptidergic' mechanism. Combining +GB with +CAP potentiated NPP/beta-FXIIa actions further by raising E 50-fold, NE sevenfold, SBP/DBP by 55/20 mmHg and HR by 87 beats/min, strengthening the efficacy of this alternate pathway. CONCLUSIONS: The cardiovascular effects of NPP/beta-FXIIa are considerably mediated by a noncholinergic (peptidergic) ACE-regulated mechanism for sympathoadrenal catecholamine release that is enhanced by +GB and/or +CAP. Under inflammatory procoagulant conditions, endogenously produced NPP/beta-FXIIa may interfere with the antihypertensive effects of ACE inhibition therapy.

Differential clot stabilising effects of rFVIIa and rFXIII-A2 in whole blood from thrombocytopenic patients and healthy volunteers.

The haemostatic effect of recombinant activated factor VII (rFVIIa;NovoSeven) in thrombocytopenic patients has been a matter of controversy. Haemostasis by rFVIIa occurs via FVIIa-mediated thrombin generation in a platelet-dependent manner and may therefore be suboptimal in patients without functional platelets. Under such conditions, a clot-stabilizing agent, such as factor XIII (FXIII), may supplement the effect ofrFVIIa and improve haemostasis. Recombinant factor XIII (rFXIII-A2) is produced as an A2 homodimer of the FXIII A subunit and is equivalent to cellular FXIII normally found in platelets. The combined effects of rFVIIa andrFXIII-A2 were evaluated in clot lysis assays using factor XIII-deficient plasma and by whole blood thrombelastography (TEG) analysis from normal donors and thrombocytopenic stem cell transplantation patients. Clotting time was shortened by rFVIIa (0.6-10 microg/ml). rFVIIa only modestly improved anti-fibrinolysis,whereas rFXIII-A2 (0-20 microg/ml) enhanced anti-fibrinolysis without effect on clotting time. TEG analysis showed rFVIIa shortened the clotting time, and enhanced clot development, maximal mechanical strength and resistance to fibrinolysis, whereas, rFXIII-A2 enhanced clot development,maximal mechanical strength and markedly enhanced resistance to fibrinolysis. These data illustrate that rFVIIa and rFXIII-A2 contribute to clot formation and stability by different mechanisms suggesting enhanced haemostatic efficacy by combining these agents.

Hemostatic and neuroprotective effects of human recombinant activated factor VII therapy after traumatic brain injury in pigs.

Human recombinant activated factor-VII (rFVIIa) has been used successfully in the treatment of spontaneous intracerebral hemorrhage. In addition, there is increasing interest in its use to treat uncontrolled bleeding of other origins, including trauma. The aim of this study was to evaluate the safety and potential effectiveness of rFVIIa to mitigate bleeding using a clinically relevant model of traumatic brain injury (TBI) in the pig. A double injury model was chosen consisting of (1) an expanding cerebral contusion induced by the application of negative pressure to the exposed cortical surface and (2) a rapid rotational acceleration of the head to induce diffuse axonal injury (DAI). Injuries were performed on 10 anesthetized pigs. Five minutes after injury, 720 microg/kg rFVIIa (n=5) or vehicle control (n=5) was administered intravenously. Magnetic resonance imaging (MRI) studies were performed within 30 min and at 3 days post-TBI to determine the temporal expansion of the cerebral contusion. Euthanasia and histopathologic analysis were performed at day 3. This included observations for hippocampal neuronal degeneration, axonal pathology and microclot formation. The expansion of contusion volume over the 3 days post-injury period was reduced significantly in animals treated with rFVIIa compared to vehicle controls. Surprisingly, immunohistochemical analysis demonstrated that the number of dead/dying hippocampal neurons and axonal pathology was reduced substantially by rFVIIa treatment compared to vehicle. In addition, there was no difference in the extent of microthrombi between groups. rFVIIa treatment after TBI in the pig reduced expansion of hemorrhagic cerebral contusion volume without exacerbating the severity of microclot formation. Finally, rFVIIa treatment provided a surprising neuroprotective effect by reducing hippocampal neuron degeneration as well as the extent of DAI.

Impairment of the hemostatic potential of platelets during storage as evaluated by flow cytometry, thrombin generation, and thrombelastography under conditions promoting formation of coated platelets.

BACKGROUND: The increasing demand for platelet (PLT) transfusions has focused attention on appropriate use. Coated PLTs are a subpopulation of highly procoagulant PLTs formed by simultaneous stimulation by the agonist's collagen and thrombin hypothesized to drive clot formation at the site of vascular injury. Prolonged storage of PLTs may reduce their ability to support optimal hemostasis upon transfusion. STUDY DESIGN AND METHODS: PLT concentrates (PCs) stored for 1, 4, 6, and 8 days were costimulated with thrombin and the collagen glycoprotein VI (GPVI) receptor agonist convulxin, and their ability to form coated PLTs was determined by flow cytometry. Further, a plasma-based thrombin generation assay and thrombelastography were used to evaluate the aged PCs' capacity to support thrombin generation and clot formation, respectively. The stored PCs were additionally tested by standard quality control methods. RESULTS: PLT quality as measured by standard analyses was acceptable according to current practice. The hemostatic potential, however, was impaired with increasing storage time. The formation of coated PLTs decreased significantly from approximately 85 to 55 percent with increasing storage time (p<0.05). The velocity of clot formation was significantly increased from Day 4 (p<0.05). The velocity of thrombin generation and resistance against fibrinolysis were significantly reduced on Day 8 compared to Day 1 of storage (p<0.05). CONCLUSION: Data in the present study suggest that storage significantly reduced the stored PLTs' ability to respond to conditions expected to exist at the site of vascular injury and that storage-induced reduction in PLT activation sensitivity correlated with a loss of hemostatic potential.

Recombinant FVIIa decreases perioperative blood transfusion requirement in burn patients undergoing excision and skin grafting--results of a single centre pilot study.

BACKGROUND: Excision of burn wounds is frequently associated with a large volume of blood loss requiring allogeneic blood transfusion. We conducted a pilot study to investigate the effect of activated recombinant coagulation factor VII (rFVIIa) on the reduction of blood transfusion requirements in burn patients undergoing excision and skin grafting. METHODS: Eighteen consecutive patients scheduled for the surgery were randomised to receive either placebo or 40 microg/kg rFVIIa administered at first skin incision, and a second dose (40 microg/kg) at 90 min later. Blood transfusion requirements during, and up to 24h post-surgery per patient and percentage full thickness wound excised were compared. In addition, postoperative complications commonly seen in patients with burns as well as adverse events related to rFVIIa were monitored. RESULTS: rFVIIa significantly decreased the total number of units of blood components transfused per patient and percentage full thickness burn wound excised compared with placebo (0.9 versus 2.2, p=0.0013) including significant fewer red blood cell units (0.5 versus 1.1, p=0.004). We further observed a trend towards improved graft survival (p=0.1) and a reduction in multiple organ failures (p=0.08) in the rFVIIa-treated group. There were no adverse events, in particular thromboembolic events. CONCLUSION: rFVIIa might be useful in decreasing blood transfusion requirements in burn patients undergoing excision and skin grafting.

Clot stabilization for the prevention of bleeding.

Coagulation is a finely tuned sequence of reactions beginning with the interaction between tissue factor (TF) and its substrate, factor VII (FVII), and resulting in the formation of a fibrin clot localized to the site of vascular endothelial disruption. While important for fibrin clot formation, thrombin also plays a role in stabilizing the clot against premature fibrinolysis by activating thrombin activatable fibrinolysis inhibitor (TAFI) and factor XIII (FXIII), the terminal enzyme in the coagulation cascade. Despite use of antifibrinolytic agents in various types of surgery to inhibit clot lysis. thereby limiting blood loss and patient exposure to allogeneic blood products, numerous patients still require transfusions for nonsurgical bleeding. This article describes new concepts of localized hemostasis, a potential role for clot stabilization, and inhibition of fibrinolysis for control of bleeding.

Study on biological variation of haemostatic parameters in clinically healthy dogs.

Thromboelastography (TEG) may be a valuable supplement to the coagulation assays activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), fibrinogen, antithrombin (AT) and D-Dimer currently used in most clinical pathology laboratories. Allowable imprecision and bias reference limits for analytical tests can be calculated based on measurements of biological variation. No studies to date have examined the effect of biological variation on these haemostasis parameters in the same group of dogs. Plasma samples were collected after a set protocol once weekly for five consecutive weeks from eight healthy dogs (four males and four females) and stored at -80 degrees C until analysis. Randomized duplicate coagulation tests and TEG analyses were performed on all plasma samples within one run. The data were analyzed for outliers and subsequently subjected to nested analysis of variance to obtain the coefficient of analytical, intra-individual and inter-individual variation. From these objective analytical performance standards for imprecision, critical difference, total error and the index of individuality were calculated to assess the utility of conventional population-based reference ranges. All the clotting times (aPTT, PT and TT), fibrinogen, AT and D-Dimer showed a degree of individuality, which may make the use of population-based reference ranges alone an insensitive interpretation criterion, whereas a population-based reference interval seems to be sensitive for interpreting all TEG parameters. Analytical performance standards for imprecision were only met for one of the coagulation assays, whereas all TEG parameters except the alpha angle, alpha achieved this analytical goal.

Validation of human recombinant tissue factor-activated thromboelastography on citrated whole blood from clinically healthy dogs.

BACKGROUND: Thromboelastography (TEG) is an analytical method that enables global assessment of hemostatic function in whole blood (WB) with evaluation of both plasma and cellular components of hemostasis. TEG has a largely unused potential in the diagnostic workup and monitoring of dogs with hemostatic disorders and it may be a valuable supplement to traditional coagulation parameters. OBJECTIVES: The objective of this study was to establish a clinically applicable reference interval for a TEG assay using recombinant human tissue factor (TF) as the activator on citrated WB from clinically healthy dogs and to evaluate the stability of citrated WB stored for 30 minutes (T30) and 120 minutes (T120) at room temperature (RT). Additionally, we evaluated the analytical variation in reaction time (R), clotting time (K), angle (alpha), and maximum amplitude (MA). METHODS: Blood was collected from 18 clinically healthy dogs. Duplicate TEG analyses with TF as the activator at a concentration of 1:50,000 were performed on canine citrated WB at T30 and T120. R, K, a, and MAwere analyzed. RESULTS: Mean TEG values at T30/T120 were R = 5.61/4.91 minutes, K = 4.20/3.34 minutes, alpha = 45.33/50.90 degrees , and MA = 47.96/50.19 mm. Significant differences in these values were observed after storage for T30 and T120 at RT, with a tendency towards hypercoagulability at T120. The mean coefficients of variation were low. CONCLUSIONS: Canine citrated WB can be used for TEG analysis with human recombinant TF as the activator when stored at RT for T30 or T120. At both time points, the analytical variation was low, suggesting that TEG analysis may be of value in evaluating dogs with hemostatic disorders. A fixed time point should be chosen for serial measurements.

Preclinical trauma studies of recombinant factor VIIa.

Preclinical studies in animals and ex vivo human blood have provided a solid rationale for conducting prospective randomized trials in trauma patients. Small animal models have been utilized to study the efficacy of recombinant activated factor VII (rFVIIa; NovoSeven) in treating thrombocytopenic rabbits and for the reversal of anticoagulation. Safety models in the rabbit also exist to test for systemic activation of clotting and pathologic thrombosis. Animal models simulating traumatic injuries in humans have primarily been performed in pigs because of species similarities in terms of coagulation characteristics and the larger internal organs. The pig studies, utilizing human rFVIIa, have shown increased strength of clot formation, decreased bleeding, and improved survival. However, these findings are not uniform and are dependant on the model chosen. All of the animal models described have provided good safety data and suggest that the use of rFVIIa is not associated with systemic activation of coagulation or microthrombosis of end organs.

Possible role of new pressor protein in hypertensive anephric hemodialysis patients.

Unexplained hypertension was observed in three anephric children on hemodialysis. We investigated the possible involvement of a novel hypertensive extra-renal enzyme new pressor protein (NPP), related to coagulation beta-FXIIa. Currently, NPP activity can only be determined by a rat bioassay model. On study day 1, pre dialysis, patients 1, 2, and 3 were hypertensive and their plasmas raised rat systolic blood pressure (SBP) by 45, 34, and 9 mmHg, respectively. Post dialysis, patients 1 and 2 reached their estimated dry body weight and their systemic pressures dropped, while patient 3 remained hypertensive and hypervolemic. Their post-dialysis plasmas raised rat SBP by 22, 14, and 9 mmHg, respectively. On day 2, similar relationships between patient SBP, volume status, and plasma NPP-like activity in rats were observed. The characteristic rat BP responses, lack of inhibition by captopril (ruling out a renin-mediated effect), and inhibition by soybean trypsin inhibitor support co-identity with NPP. Plasma FXIIa (combined alpha-FXIIa and beta-FXIIa) was measured by immunoassay and found to be elevated in all patients. This investigation suggests that there is high endogenous NPP activity in the plasmas of these hypertensive hemodialysis patients, it changes with SBP and fluid volume, and is a possible contributor to their hypertension. Further studies are required to examine the wider applicability of these novel findings.

Improved hemostasis with superactive analogs of factor VIIa in a mouse model of hemophilia A.

It is currently debated whether the mechanism of action of therapeutic doses of recombinant factor VIIa (rFVIIa, Novo-Seven) relies on the tissue factor (TF)-independent activity of the enzyme. The present study was conducted to investigate the in vivo hemostatic effects of rFVIIa and 3 analogs thereof with superior intrinsic activity (FVIIaIIa, K337A-FVIIaIia, and M298Q-FVIIa) in mice with antibody-induced hemophilia A. A highly significant dose response was observed for the bleeding time and blood loss for each of the rFVIIa variants. The bleeding time and blood loss were normalized after administration of 10 mg/kg rFVIIa, 3 mg/kg K337A-FVIIaIia, and 3 mg/kg M298Q-FVIIa, indicating a potency of these FVIIa analogs 3-4 times above that of rFVIIa in FVIII-depleted mice. The different in vivo potencies of the various forms of FVIIa could not be explained by the pharmacokinetics. Histopathological evaluation of kidneys revealed no signs of treatment-related pathological changes even after treatment with the superactive variants. The fact that FVIIa analogs with enhanced intrinsic activity are more efficacious in the murine hemophilia A model strongly suggests that the TF-independent procoagulant activity of FVIIa contributes to its clinical hemostatic effect.

Recombinant factor VIIa reduces bleeding in severely thrombocytopenic rabbits.

Severe thrombocytopenia is a common complication to intensive chemotherapeutic regimens. For bleeding episodes associated with severe thrombocytopenia, the current standard treatment is platelet transfusion. However, due to several transfusion complications such as transfusion-transmitted diseases, platelet refractoriness and immunomodulation, as well as increasing problems with sufficient supply of platelet products, it is imperative to search for alternatives to platelet transfusion. To test the efficacy of recombinant activated human coagulation factor VII (rFVIIa, NovoSeven) in thrombocytopenia, a preclinical study was conducted in thrombocytopenic rabbits. Thrombocytopenia was induced by a combination of gamma-irradiation and the use of platelet antibodies, and the effect of rFVIIa on nail cuticle bleeding was determined. Administration of rFVIIa at 2 mg/kg significantly shortened the prolonged bleeding time in thrombocytopenic animals (rFVIIa vs. control, median 23 min 41 s vs. 60 min, p=0.016) as well as significantly reducing the blood loss (rFVIIa vs. control, median: 8.8 vs. 12.2 nmol hemoglobin/ml, p=0.016). This effect was also reflected by a significant reduction of the prothrombin time, activated partial thromboplastin time, as well as improvement in clotting parameters in an in vitro thromboelastography thrombocytopenia model. Histopathological evaluation of kidney biopsies for the presence of micro thrombi did not reveal evidence of prothrombotic effects of rFVIIa in this model. These data demonstrate the haemostatic efficacy of rFVIIa in a rabbit model of severe thrombocytopenia. Clinical trials will be needed to further explore the potential of NovoSeven as a haemostatic agent in thrombocytopenic patients.