A lyophilized human reference plasma for coagulation factors. Evidence for stability of factors I, II, V, and VII through XII.

Results are reported of a one-year study on the stability of a lyophilized normal human reference plasma, originally standardized for eight clotting factor activities against a freshly prepared pool of citrated, platelet-poor normal human plasma. The stability of any particular coagulant activity in this reagent was represented mathematically as a function of the observed clotting times obtained under specific assay conditions, where any observed change in the clotting times over the trial period was taken to reflect a change in the stability of the reference plasma. Taking into consideration estimates of variability contributed by different donors of factor-deficient substrata-plasma, different lots of substrate from the same donor, different technologists, and certain age-of-substrate lot trends, there was no statistically significant change in functional activity for Factors V and VII through XII in the reagent when it was stored unreconstituted below -25C. By less rigorous criteria, both clottable fibrinogen (Factor I) and the thrombin-forming potential of prothrombin (Factor II) also were stable during this same period. Based on these results, it was concluded that when suitably prepared, lyophilized human plasma can be used as a stable, secondary standard reference for the assay of coagulation factors.

Association of factor V activity with membranous vesicles released from human platelets: requirement for platelet stimulation.

The membrane-associated factor V-like activity (platelet factor 1, PF1) and the phospholipid-like catalytic surface activity (platelet factor 3, PF3) were studied in human platelets from normal and two factor V-deficient donors. Collagen stimulation or mechanical disruption of gel-filtered platelets was necessary for the expression of significant amounts of PF1 and PF3. Stimulation was also necessary for the uptake of factor V or Va by PF1-deficient platelets from the factor V-deficient donors. The activity of PF1 was also generated by association of factor V or Va with membrane-rich fractions obtained by gel filtration of the supernatant from collagen-stimulated or frozen-thawed PF1-deficient platelets. The amount of PF1 obtained by such all-or-none binding experiments was directly proportional to the amount of PF3 already expressed in the platelet preparation. These data have been summarized in terms of a hypothesis which views PF1 and PF3 to be activities associated with membranous vesicles released from platelets only after stimulation.

Comparison of the abilities of synthetic and platelet-derived membranes to enhance thrombin formation.

The relative abilities of platelet-derived membranes and synthetic phospholipid vesicles to enhance the prothrombinase-catalyzed conversion of prothrombin to thrombin have been determined. For each type of membrane, the maximum amount of thrombin formed as a function of amount of available lipid was measured using a chromogenic substrate assay. The lipid concentration at which the amount of thrombin formed began to exceed that formed in the absence of lipid (critical phospholipid concentration) was used to compare the surfaces' abilities to support thrombin formation. For platelet-derived membranes and for equimolar, charged-lipid/phosphatidylcholine (PC) vesicles, the critical concentrations increased in the following order: platelet-derived membranes approximately equal to phosphatidylserine (PS) approximately equal to phosphatidic acid (PA) less than monomethyl PA and monoethyl PA much less than phosphatidylinositol and phosphatidylglycerol. For mixed anionic/neutral lipid vesicles above their phase transitions, measured critical concentrations were relatively insensitive to changes in lipid acyl chains, the neutral lipid component, and membrane curvature but were sensitive to changes in the anionic lipid content of the mixtures. Comparison of these data suggested that equimolar PS/PC and PA/PC vesicles can emulate reasonably well the thrombin-generating ability of platelet-derived membranes.

Expression of coagulant activity in human platelets: release of membranous vesicles providing platelet factor 1 and platelet factor 3.

The relationship between the appearance of membrane-associated factor V-like activity (platelet factor 1, PF1) and phospholipid-like catalytic activity (platelet factor 3, PF3) has been examined, in vitro, in collagen-stimulated, human platelets. Both activities increased 7 fold upon collagen treatment relative to stirred controls. After sedimentation of stimulated platelets, 31% of total PF1 and 41% of PF3 remained in the supernatant fraction. PF1 eluted from a Sepharose CL-4B column in the same void volume fractions as PF3, phospholipid, and vesicular particles. These fractions had roughly 100 fold (lipid basis) or 1000 fold (protein basis) enhanced specific activity when compared to the stimulated platelet suspension. Freeze-fracture electron microscopy demonstrated that these void volume fractions contain two populations of membranous vesicles (80-200 nm and 400-600 nm in diameter). Upon centrifugation of the void volume fractions, PF1 and PF3 activities, phosphate-containing material, and ultraviolet-absorbing material all sedimented at the same rate, indicating that PF1 and PF3 are activities associated with one or both of the platelet-derived vesicle populations. Finally, we examined the effects of inhibitors on the appearance of PF1, PF3, platelet factor 4, total intrinsic factor V activity, and serotonin as well as on platelet aggregation. These studies suggest that the collagen-stimulated release of PF1 and PF3 is not coupled to either platelet aggregation or PF4 release but is probably a separate phase of the release reaction.

Phospholipid vesicle stimulation of proacrosin activation.

Aqueous dispersions of synthetic phospholipids, in the form of anionic, single bilayer vesicles, were observed to stimulate the appearance of acrosin esterase activity from its zymogen precursor, proacrosin. Enzymatic activity measurements, in parallel with polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate, indicated that the enzymatic activity produced had resulted from the conversion of proacrosin to acrosin (EC 3.4.21.10), and not from the direct stimulation of a possible proacrosin esterase activity. It is suggested that such bilayer lipid vesicles can be used as a model membrane system to study the activation of proacrosin in vitro.

Coagulation changes in elective surgery and trauma.

Although antithrombin-3 (AT-3), a naturally-occurring inhibitor of thrombin, has been associated with a variety of thrombotic disorders, it has been studied in surgery and trauma. Three groups of patients were studied: Group I (20 patients) who underwent elective surgery; Group II (ten patients) who sustained moderate trauma: Group III (ten patients) who sustained severe trauma. Hypercoagulability panels were run preoperatively, intraoperatively, and postoperatively. Nine units of banked blood were also tested. The coagulation pattern changed during the stress, becoming hypercoagulable in proportion to the stress endured by the patient. In the severe trauma group, AT-3 fell significantly (p less than or equal to 0.002) in all patients, indicating extreme hypercoagulability. Three of these patients sustained thrombosis and loss of the involved extremity. The banked blood was found to be hypercoagulable. It appears that patients who sustain severe trauma, have multiple transfusions, and major operative procedures are at increased risk of developing postoperative thrombotic complications, including loss of limb.

Phase behavior of mixed phosphatidylglycerol/phosphatidylcholine multilamellar and unilamellar vesicles.

The phase behavior of dipentadecanoylphosphatidylglycerol (DC15PG)/dimyristoylphosphatidylcholine (DMPC) mixtures has been studied in both small, unilamellar vesicles and large, multilamellar vesicles. We have used both the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and high-sensitivity differential scanning calorimetry to detect temperature-dependent changes in membrane structure. Electron microscopy has demonstrated different fracture face morphologies for large, multilamellar vesicles depending on sample composition and temperature. These data have been interpreted in terms of proposed phase diagrams for this lipid mixture. The shapes of the proposed phase diagrams have led us to conclude that DMPC and DC15PG mix freely in the plane of a lipid bilayer only at less than 50 mol % DC15PG. At higher DC15PG content, the data have been interpreted as reflecting substantial compositional inhomogeneities in the plane of the bilayer, if not phase immiscibility, even in the fluid phase. In addition, small vesicles containing greater than 50 mol % DC15PG were unstable in the ordered phase and spontaneously converted to larger vesicles. Finally, the anisotropy of DPH fluorescence was found to be invariant with DC15PG content at temperatures just above the liquidus phase line in small, unilamellar vesicles. This demonstrated that inclusion of negatively charged phosphatidylglycerol does not noticeably affect the order within the acyl chain region of the bilayer, relative to phosphatidylcholine.

Calcium-dependent and calcium-independent interactions of prothrombin fragment 1 with phosphatidylglycerol/phosphatidylcholine unilamellar vesicles.

We have measured the phase behavior of mixed dipentadecanoylphosphatidylglycerol (DC15PG)/dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUV) in the presence of saturating (greater than 98% occupancy of binding sites) concentrations of bovine prothrombin fragment 1 and 5 mM Ca2+. Binding of fragment 1 in the presence of Ca2+ was verified by an increase in 90 degrees light scattering. Only in the cases of DC15PG/DMPC SUV below their phase transition and of pure DMPC SUV were such light scattering measurements not reversible upon addition of ethylenediaminetetraacetic acid to complex Ca2+. Phase-behavior changes of DC15PG/DMPC SUV as monitored by diphenylhexatriene fluorescence anisotropy occurred in concert with the binding of fragment 1. The major effects of peptide binding on SUV phase behavior were to raise the phase-transition temperature by 2-15 degrees C, depending on vesicle composition, and, in general, to make the phase diagram for these small vesicles closely resemble that of large vesicles. No evidence was obtained for the existence of lateral membrane domains with distinct compositions induced by the binding of prothrombin fragment 1 plus Ca2+. Surprisingly, fragment 1 without Ca2+ also altered the phase behavior of DC15PG/DMPC SUV. Most striking was the effect of fragment 1 (with or without Ca2+) on DMPC SUV phase behavior. Freeze-fracture electron microscopy demonstrated that pure DMPC vesicles were induced to fuse in the presence of fragment 1, while vesicles containing DC15PG remained intact. The rate of DMPC SUV fusion (followed by 90 degrees light scattering) increased with increasing fragment 1 concentration but was not saturable up to 40 microM fragment 1, suggesting a weak, nonspecific interaction between fragment 1 and the neutral phospholipid vesicle.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparin cofactor activities in a family with hereditary antithrombin III deficiency: evidence for a second heparin cofactor in human plasma.

Plasma levels of antithrombin-heparin cofactor, determined by heparin-dependent antithrombin assay, and antithrombin III antigen were measured in 22 members of a large kindred predisposed to venous thrombosis. While 11 members had reduced plasma levels of both antithrombin-heparin cofactor and antithrombin III antigen, the levels of antithrombin-heparin cofactor were always greater than the levels of antithrombin III antigen: 66% (+/- 7%) and 49% (+/- 5%) of normal plasma, respectively. Pooled normal plasma and plasma from one of the affected family members (60% antithrombin-heparin cofactor and 47% antithrombin III antigen) were fractionated by heparin-agarose affinity chromatography. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 0.4 M NaCl and did not cross-react with antibody specific for antithrombin III and did not inhibit factor Xa at an appreciable rate in the presence of heparin, was designated heparin cofactor A. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 2.0 M NaCl, was functionally and antigenically identified as antithrombin III. The concentrations of heparin cofactor A in normal and patient plasma were similar (4.5 x 10(-7) M), while the concentration of antithrombin III in patient plasma (8.0 x 10(-7) M) was only 50% of normal (1.6 x 10(-6) M). The functional properties of both heparin cofactor A and antithrombin III obtained from patient plasma were normal. From the results of the present study it would appear that the antithrombin-heparin cofactor concentrating measured in patient plasma reflects the combined concentrations of heparin cofactor A and antithrombin III. Since heparin cofactor A does not cross-react with antibody to antithrombin III, the concentration of antithrombin III antigen in patient plasma is thus lower than the concentration of antithrombin-heparin cofactor.

Monoclonal antibodies specific for prothrombin fragment 1.2 and their use in a quantitative enzyme-linked immunosorbent assay.

Prothrombin fragment 1.2 (F1.2) is an activation peptide generated during a critical event of blood coagulation, the conversion of prothrombin to thrombin. As a marker of thrombin generation, F1.2 has clinical potential in assessing thrombotic risk and monitoring anticoagulant therapy. In developing a highly specific, monoclonal antibody-based immunoassay of human plasma F1.2, we generated six murine anti-F1.2 monoclonal antibodies, using as immunogen a synthetic peptide (sequence: CGSD-RAIEGR) similar to the unique carboxyl terminus of F1.2. Each antibody bound F1.2 but not prothrombin. Epitope mapping studies with one antibody (5-3B) showed that optimum binding required six to eight amino acids plus a terminal arginine to emulate the F1.2 carboxyl terminus. A quantitative sandwich ELISA for human plasma F1.2 was configured with monoclonal antibody 5-3B as the capture antibody and peroxidase-labeled polyclonal antibodies to the F1.2 amino-terminal region as detector antibodies. Calibrators were prepared by adding purified F1.2, 0-10 nmol/L, to F1.2-depleted plasma. Assay characteristics included the following: mean (+/- SD) analytical recovery of 98% +/- 13%; no interference from lipemia, hemolysis, icterus, or thrombolytic agents; 0.08 nmol/L sensitivity; and mean intra- and interassay imprecision (three lots) < 12% at both low and high concentrations of F1.2.