Recombinant coagulation factor VIIa (FVIIa) is established as a useful therapy for enhancing hemostasis in hemophiliacs. Our data suggest that it acts by binding to activated platelets and activating FXa on the platelet surface, independent of its usual cofactor, tissue factor (TF). 1,2 The activation of FX on the platelet surface would normally be via the FIXa/VIIIa complex, which is deficient in hemophiliacs. Platelet FXa produces the burst of thrombin generation required for effective clot formation. Thus, at high doses, FVIIa can partially restore platelet surface FX activation and thrombin generation.
Recently, high-dose FVIIa has also been used to manage bleeding complicating trauma or surgery. 3,4 Although no controlled trials have been completed, it appears to be useful in patients who develop diffuse hemorrhage, because of a coagulopathy that cannot be managed by maximal transfusion therapy. High-dose FVIIa has also been shown to reduce blood loss and prolong survival in a hypothermic pig model of hepatic trauma. 5 However, some patients do not respond to high-dose FVIIa. Therefore, we wanted to determine whether two potential complications of surgery and trauma, hypothermia and acidosis, might impair the effectiveness of FVIIa as a hemostatic agent.
Hypothermia associated with severe trauma is correlated with a significantly worse prognosis than either trauma or hypothermia alone. Isolated hypothermia of 32.2°C leads to a 23% mortality rate, whereas trauma-induced hypothermia less than 32°C is associated with 100% mortality. 6–8 The primary risk of hypothermia is abnormal bleeding. Several factors have been proposed to contribute to this coagulopathy, including reduced activity of coagulation enzymes and platelets, activation of fibrinolysis, and endothelial injury (reviewed in Kirkpatrick et al. 9).
Acidosis is also associated with worse survival in trauma and surgery patients. It can result from metabolic derangements that develop in the sickest patients. Excess lactic acid production associated with tissue hypoxia is the best recognized cause. Lactic acid is the end product of anaerobic metabolism, and its level is related to oxygen availability. Acidosis can impair coagulation and worsen the risk of serious hemorrhage. 10 Massive transfusion can exacerbate acidosis, because stored blood has a reduced pH.
Thus, if FVIIa is used to manage bleeding in surgical and trauma patients, some significant number of them are likely to suffer from hypothermia, acidosis, or both. The goal of the current study was to examine the effect of temperature and pH on the TF-dependent and -independent activity of FVIIa, and on the ultimate complex in the coagulation pathway, the FXa/FVa (prothrombinase) complex.
MATERIALS AND METHODS
Spectrozyme FXa was obtained from American Diagnostica (Greenwich, CT) and Chromozym Th was obtained from Boehringer Mannheim (Germany). Phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were obtained from Avanti Polar Lipids (Birmingham, AL). Phospholipid vesicles (15% PS/41% PC/44% PE) were prepared as described. 11 Briefly, PS, PC, and PE were combined, dried under nitrogen gas, and resuspended in cyclohexane. The resuspended lipids were lyophilized, resuspended in HEPES-buffered saline (pH 7.4) containing 0.1 mmol/L EDTA, and extruded through a 0.2-μm filter (Gelman) 10 times. Lipid vesicles were stored under nitrogen.
Prothrombin and FIX were purified as described. 12,13 FX was purchased from Enzyme Research Laboratories (South Bend, IN) and repurified on Q Sepharose with CaCl2 elution. 14 FVa was obtained from Hematologic Technologies. Recombinant human TF prothrombin time reagent (Innovin) was obtained from Dade-Behring (Miami, FL). FVIIa and thrombin were the generous gifts of Dr. Ulla Hedner (Novo Nordisk, Denmark) and Dr. Herbert Whinna, 15 respectively.
Blood was collected from healthy, consenting adult volunteers (aged 21–56 years old) under protocols approved by the Duke University and Durham VA Medical Center Institutional Review Boards. Platelets were freshly isolated from individual donors by density gradient centrifugation and gel filtration as described. 1 Briefly, prostaglandin E1 (5 μg/mL, final concentration) was added to citrated blood that was centrifuged through Accuprep Lymphocyte gradient medium. The mononuclear band was diluted with an equal volume of 1× CGS buffer (13 mmol/L citrate, pH 7.4, 123 mmol/L NaCl, 33 mmol/L dextrose, final concentration) containing prostaglandin E1 (5 μg/mL, final concentration) and centrifuged at 800 rpm for 10 minutes. The platelet-containing supernatant was then gel filtered over Agarose C4BL in Tyrodes buffer (15 mmol/L HEPES, 3.3 mmol/L NaH2PO4 [pH 7.4], 138 mmol/L NaCl, 2.7 mmol/L KCI, 1 mmol/L MgCI2, 5.5 mmol/L dextrose) containing 1 mg/mL bovine serum albumin. Platelets were kept at 37°C until used.
FVIIa/TF Activity Assay
The activity of FVIIa in complex with TF was measured as factor X activation. 16 Recombinant human TF was reconstituted as directed by the manufacturer; then, a 1:250 dilution was mixed with FVIIa (200 pmol/L, final concentration) and FX (135 nmol/L, final concentration) in 3 mmol/L CaCl2. For the temperature studies, the reactions were kept at 24°, 33°, or 37°C. For the pH studies, the pH of the reaction was adjusted by combining solutions of saline buffered with 20 mmol/L MES or HEPES to obtain the desired pH. The reactants were then diluted in buffer of the appropriate pH. At timed intervals, aliquots were transferred into wells containing EDTA (5 mmol/L, final concentration) to chelate the calcium and stop further activation of FX. FXa activity was measured by adding the samples to the chromogenic substrate Spectrozyme FXa (600 μmol/L, final concentration), and monitoring the increase in absorbance at 405 nm in a plate-reading spectrophotometer (SPECTRAmax Microplate Spectrophotometer 190, Molecular Devices, Sunnyvale, CA).
FVIIa Activity in the Absence of TF on Phospholipid Vesicles and Platelets
FVIIa activity on phospholipid vesicles in the absence of its cofactor was also measured by FXa generation. Briefly, 60 μmol/L phospholipid vesicles was combined with a solution of FVIIa (100 nmol/L, final concentration) and FX (135 nmol/L, final concentration) at 24°, 33°, or 37°C. For the pH studies, the pH of the reaction was adjusted by combining solutions of saline buffered with 20 mmol/L MES or HEPES. Aliquots (25 μL) from each experimental condition were removed into wells containing EDTA (5 mmol/L, final concentration). After all the samples had been taken, Spectrozyme FXa was added to each sample well and the increase in absorbance at 405 nm monitored as above.
FVIIa activity on activated platelets was measured by allowing FVIIa (100 nmol/L) to activate FX (135 nmol/L) on the platelet surface at the desired temperature. Aliquots were transferred into a solution containing prothrombin (1.4 μmol/L) and Chromozyme Th (500 μmol/L, final concentration) at room temperature. The FXa formed on the platelet surface was then detected by its ability to activate prothrombin to thrombin, which subsequently cleaved the chromogenic substrate. The increase in absorbance at 405 nm was monitored in a plate-reading spectrophotometer.
Activity of the FXa/FVa Complex on Platelets and Phospholipid Vesicles
The activity of the FXa/FVa complex on phospholipid vesicles was measured as prothrombin activation. FXa (4 nmol/L), FVa (10 nmol/L), and 3 mmol/L CaCl2 were added to a solution of 60 μmol/L phospholipid at the desired temperature or pH. Prothrombin (1.4 μmol/L) was then added to each sample, and timed aliquots were removed and assayed for thrombin generation by measuring cleavage of the chromogenic substrate, Chromozyme Th (500 μmol/L).
The activity of the FXa/FVa complex on platelets was measured as follows. Freshly isolated platelets (50–150 × 103/μL) were activated with 1.0 nmol/L thrombin at 37°C for 10 minutes in the presence of 4 nmol/L factor Xa and 3 mmol/L CaCl2. Aliquots of the activated platelets were then equilibrated to 24°, 33°, or 37°C for 5 minutes. As above, prothrombin was added to each tube, and timed aliquots were removed and assayed for thrombin generation by cleavage of the chromogenic substrate, Chromozym Th.
Data were processed and analyzed using Microsoft Excel. Comparison of each temperature group was carried out by normalizing values from each experiment to 37°C and comparing the means using a nonpaired t test (Fig. 1). Each point in the temperature data is the mean ± SD of three separate experiments. In experiments where platelets were used, the data are from platelets from three different normal donors.
Data on the effect of pH on the activity of FVIIa on phospholipid, FVIIa/TF, and FXa/FVa were normalized so that the rate of activation of the substrate zymogen was set equal to 1 at pH 7.4. The rates at other pH values are expressed relative to this rate (Figs. 2 and 3). The rate at pH = 7.4 was compared with the rate at each lower pH value using Tukey’s ω-procedure for multiple comparisons. The pH data represent the mean ± SD of four separate experiments. In experiments where platelets were used, the data are from platelets from four different donors.
Decreased Temperature Decreases the Activity of FVIIa in Complex with TF but Increases the Activity of FVIIa Alone on a Phospholipid Surface
The activity of the FVIIa/TF complex was, as expected, directly dependent on temperature. The rate of FXa generation was reduced 2.6- and 1.1-fold at 23° and 33°C, respectively, compared with 37°C (Fig. 1). This is consistent with the expected twofold increase in rate for every 10°C increase in reaction temperature for most enzymes. 17 In marked contrast, the rate of FX activation by FVIIa alone did not decrease, but actually increased as the temperature was decreased. This effect was seen for FVIIa activation of FX on synthetic phospholipid vesicles (Fig. 1) and on activated platelets. The relative rate of activity for FVIIa/phospholipid vesicles was significantly higher (p < 0.05) than that of FVIIa/TF and FXa/Va at both 23° and 33°C.
Decreased Temperature Decreases the Activity of the FXa/Va Complex
The activity of the FXa/FVa (prothrombinase) complex was also determined as a function of temperature for comparison with FVIIa. Prothrombinase activity decreased in a temperature-dependent fashion from 37° to 33° to 24°C (Fig. 1). Similar to the activity of the FVIIa/TF complex, the rate of thrombin generation was consistent with the expected twofold increase in rate for every 10°C increase in reaction temperature. 17
Decreased pH Decreases the Activity of FVIIa Alone on Phospholipid Surface and Also in Complex with TF
The activity of FVIIa on a phospholipid surface was reduced by over 90% at a pH of 7.0 compared with the rate at 7.4 (p < 0.05). The activity of the FVIIa/TF complex was reduced to a somewhat lesser degree as the pH decreased, by 55% at pH 7.0 compared with 7.4 (Fig. 2), but the reduction was still statistically significant (p < 0.05). The activity of FVIIa on platelets was not determined, because the alteration in pH seemed to alter platelet function, including release and/or activation of FV(a) from platelet stores (data not shown). Thus, we could not easily isolate the effect of pH on FVIIa or FXa/FVa activity on platelet surfaces.
Decreased pH Decreases the Activity of the FXa/FVa Complex
Similar to the results obtained for FVIIa and FVIIa/TF, the rate of prothrombin activation by FXa/FVa complexes on phospholipid vesicles was dramatically reduced as the pH was reduced (Fig. 3). The activity of the prothrombinase complex was reduced by approximately 70% at pH 7.0 compared with 7.4 (p < 0.05).
Our results demonstrate that a clinically significant decrease in temperature does not reduce the TF-independent activity of FVIIa, the activity we hypothesize to be primarily responsible for its therapeutic effect. 2 By contrast, a reduction in pH from 7.4 to 7.0 nearly abolished FVIIa activity and severely reduced the activity of the FXa/FVa complex that activates prothrombin to thrombin. Thus, we conclude that hypothermic patients are likely to benefit from FVIIa therapy, whereas acidotic patients may not.
Our data showed a surprising effect of temperature on FVIIa activity. In general, one expects that the activity of an enzyme will double for every 10°C that the temperature is increased—at least until the temperature is so high that the enzyme begins to denature. This was exactly what we saw when we examined the activity of the FVIIa/TF and FXa/FVa complexes. However, the activity of FVIIa in the absence of its cofactor did not decrease as the temperature declined between 37° and 24°C. In fact, the activity actually increased. This finding is consistent with the work of Petersen et al., 18 who studied FVIIa cleavage of a chromogenic substrate in solution. They postulated that when FVIIa was not stabilized by its cofactor, TF, increasing the temperature caused it to be converted to an inactive (or less active) form. Thus, our data and those in the literature suggest that if FVIIa is acting on platelet surfaces in patients, it should work at least as well under hypothermic conditions as at 37°C.
By contrast to the temperature data, we found that a decrease in the pH profoundly impaired the activity of FVIIa alone and the FVIIa/TF and FVa/Xa complexes. It is known that the coagulation proteases have pH optima well above the physiologic range. Our data are consistent with the published literature and suggest that hemostasis is likely to be profoundly impaired in acidotic patients, because the activity of the proteases in the coagulation system is dramatically reduced by even a small decrease in pH. Therapeutically administered FVIIa may not be able to overcome the hemostatic defect in such patients.
High-dose FVIIa therapy appears to have potential for treating bleeding complicating trauma or surgery in patients with no preexisting coagulation disorder. FVIIa therapy has been used primarily in cases where surgical repair and transfusion therapy have failed to control bleeding. FVIIa has been reported to effectively control hemorrhage in cases of gunshot wounds, liver trauma, intra-abdominal hemorrhage, and gastrointestinal bleeding. 19–22 Such patients are often acidotic and hypothermic and develop a coagulopathy that leads to diffuse microvascular bleeding. The presence of this diffuse oozing makes surgical repair of anatomic lesions difficult. It also interferes with the use of topical agents, such as fibrin glue, because these agents are much more effective when applied to a relatively dry field. Thus, it appears that high-dose FVIIa therapy may be able to improve hemostasis in patients for whom few other treatment options are available.
Recombinant FVIIa has been used extensively and is recognized to be very effective in establishing hemostasis in hemophiliacs with an inhibitor. However, even in this clinical setting there is not complete agreement on the mechanism of action of high-dose FVIIa therapy. We have evidence that at levels of FVIIa required for efficacy in hemophiliacs, FVIIa binds to the surface of activated platelets. 1 The platelet-bound FVIIa activates small amounts of FX on the platelet surface. In this regard, the activated platelet surface binds FVIIa and enhances its activity in a manner similar to that of phospholipid vesicles. This activity can partially compensate for the lack of FX activation by the platelet-surface FIXa/VIIIa complex—which is deficient in hemophiliacs. Although the activity of the platelet-bound FVIIa is low relative to the FIXa/VIIIa and FVIIa/TF complexes, it is able to provide enough FXa to partially restore platelet-surface thrombin generation and support hemostatic clot formation. 2 A platelet-surface mechanism of action explains why administration of FVIIa does not lead to systemic activation of coagulation: its effects are localized to the site of injury where platelets adhere and are activated.
Although numerous case reports attest to its effectiveness in trauma patients, no controlled clinical trials have yet been conducted to examine the role of high-dose FVIIa therapy in this setting. Therefore, it is not currently clear which patients will benefit from this costly therapy, the appropriate dose of FVIIa for trauma patients, or what concurrent conditions might affect the efficacy of FVIIa. A limited number of studies in swine suggest that FVIIa can reduce hemorrhage and mortality after experimental liver trauma, even in the setting of hypothermia. 23–25 No clinical or animal studies have examined the effect of acidosis on the effectiveness of FVIIa, even though acidosis commonly develops in traumatized patients. One animal study did find that the volume of blood lost in a swine model of hemorrhagic shock was correlated with the degree of tissue acidosis that developed. The additional blood was lost because of continued oozing at sites where tissue samples were taken by biopsy and may reflect a coagulopathy directly attributable to acidosis. 26
Our current study examined the effects of pathophysiologically relevant levels of acidosis and hypothermia on the biochemical activity of FVIIa. Our results are consistent with the literature from the swine model in that hypothermia did not impair the activity of FVIIa. However, our results suggest that FVIIa may not be very effective in acidotic patients.
Our study did not address the question of whether the activity of FVIIa might be affected in vivo by pathologic changes in patients who have suffered trauma, extensive hemorrhage, or shock. For example, it is possible that platelet function might be altered in some such patients and that these alterations might impact the activity of FVIIa. The effects of altered cellular function in seriously ill patients would be superimposed on the biochemical effects of hypothermia and acidosis on the coagulation proteins. Thus, it seems likely that the efficacy of FVIIa in an individual patient might be affected by a number of variables and not only the pH and temperature.
We recommend that clinicians who use FVIIa in trauma patients take note of the level of acidosis present and consider biochemical correction of acidosis before administration of FVIIa. Because of the profound effect of pH on the prothrombinase (FXa/FVa) complex, correction of acidosis may by itself improve hemostasis. Finally, we recommend that the presence and degree of acidosis be considered in the design of clinical trials of FVIIa in postsurgical and trauma patients.
We thank Angie Lenkowski, for excellent technical assistance, and Lars-Christian Peterson, DSc, and Harold R. Roberts, MD, for helpful discussions.
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Keywords:© 2003 Lippincott Williams & Wilkins, Inc.
Hemostasis; Hemorrhage; Platelets; Factor VIIa