Quality of frozen transfusable plasma prepared from whole blood donations in Canada: an update.

BACKGROUND: Transfusable plasma is obtained by processing whole blood donations, by apheresis, or as solvent/detergent plasma (SD plasma), a pooled pathogen-reduced plasma product. The quality of plasma is typically assessed by testing the activities of multiple coagulation-related plasma proteins, due to a lack of clinical trial data linking plasma composition to clinical endpoints. We sought to update previous quality surveys of Canadian frozen plasma (FP; manufactured from single donor whole blood donation and frozen within 24h of phlebotomy), to provide transfusionists with a more complete picture of its characteristics. STUDY DESIGN AND METHODS: FP units (n=131) were tested for: the activity of factors V, VII, VIII, X, and XI, protein S (PS), α2-antiplasmin (AP), and fibrinogen; and the activated partial thromboplastin (APTT) and prothrombin (PT) times. Comparisons were made to: previous Canadian FP surveys; and to studies of single-donor plasma and SD plasma from other nations. RESULTS: Mean FVIII, fibrinogen, or APTT values did not differ from the previous annual survey of Canadian FP; FV activity was increased and PT values decreased. FP produced with or without leukoreduction differed only in mean APTT. Canadian FP exhibited generally similar quality to that reported by other organizations in Europe and Asia for similarly manufactured single-donor plasma, but contained notably higher PS and AP (≈ four-fold) activities than did SD plasma. CONCLUSION: Our results indicate that Canadian FP is of similar quality to single-donor products produced in other jurisdictions. While it is of arguably superior in vitro quality to an SD plasma product recently licensed in Canada, these differences are highly unlikely to have clinical significance for most indications for plasma transfusion.

Proteolytic modulation of factor Xa-antithrombin complex enhances fibrinolysis in plasma.

Our previous work showed that purified coagulation factor Xa (FXa) acquires fibrinolysis cofactor activity after plasmin-mediated cleavage. The predominant functional species is a non-covalent heterodimer of 33 and 13kDa, termed Xa33/13, which has predicted newly exposed C-terminal lysines that are important for tissue plasminogen activator (tPA)-mediated plasminogen activation to plasmin. To provide evidence that this mechanism occurs in a physiological context, here we demonstrated the appearance of Xa33 in clotting plasma by western blot analysis. Since the normal fate of FXa is stable association with antithrombin (AT), an AT western blot was conducted, which revealed a band of ~13kDa higher apparent molecular weight than AT that appeared concurrent to Xa33. Sequencing of purified proteins confirmed the generation of Xa13 covalently bound to AT and Xa33 (Xa33/13-AT) by cleavages at Lys-Met339 and Lys-Asp389. Ligand blots demonstrated (125)I-plasminogen binding to the Xa33 subunit of plasmin-generated Xa33/13-AT. Purified XaAT added to plasma that was induced to clot enhanced the rate of tPA-mediated fibrinolysis by ~16-fold. Similarly, purified plasminogen activation by tPA was enhanced by ~16-fold by XaAT. Plasmin cleaves XaAT and exposes plasminogen binding sites at least 10-fold faster than FXa. Here we demonstrate a novel function for AT, which accelerates the modulation of FXa into the fibrinolytic form, Xa33/13. The consequent exposure of C-terminal lysine binding sites essential for plasminogen activation enhances fibrinolysis. These results are consistent with a model where auxiliary cofactors link coagulation to fibrinolysis by priming the accelerating role of fibrin.

Enhanced fibrinolysis by proteolysed coagulation factor Xa.

We previously showed that coagulation factor Xa (FXa) enhances activation of the fibrinolysis zymogen plasminogen to plasmin by tissue plasminogen activator (tPA). Implying that proteolytic modulation occurs in situ, intact FXa (FXaalpha) must be sequentially cleaved by plasmin or autoproteolysis, producing FXabeta and Xa33/13, which acquire necessary plasminogen binding sites. The implicit function of Xa33/13 in plasmin generation has not been demonstrated, nor has FXaalpha/beta or Xa33/13 been studied in clot lysis experiments. We now report that purified Xa33/13 increases tPA-dependent plasmin generation by at least 10-fold. Western blots confirmed that in situ conversion of FXaalpha/beta to Xa33/13 correlated to enhanced plasmin generation. Chemical modification of the FXaalpha active site resulted in the proteolytic generation of a product distinct from Xa33/13 and inhibited the enhancement of plasminogen activation. Identical modification of Xa33/13 had no effect on tPA cofactor function. Due to its overwhelming concentration in the clot, fibrin is the accepted tPA cofactor. Nevertheless, at the functional level of tPA that circulates in plasma, FXaalpha/beta or Xa33/13 greatly reduced purified fibrin lysis times by as much as 7-fold. This effect was attenuated at high levels of tPA, suggesting a role when intrinsic plasmin generation is relatively low. FXaalpha/beta or Xa33/13 did not alter the apparent size of fibrin degradation products, but accelerated the initial cleavage of fibrin to fragment X, which is known to optimize the tPA cofactor activity of fibrin. Thus, coagulation FXaalpha undergoes proteolytic modulation to enhance fibrinolysis, possibly by priming the tPA cofactor function of fibrin.

Differential contributions of Glu96, Asp102 and Asp111 to coagulation factor V/Va metal ion binding and subunit stability.

Blood coagulation FV (Factor V) is activated by thrombin-mediated excision of the B domain, resulting in a non-covalent heterodimer, FVa (activated FV). Previous studies implicated Glu96, Asp102 and Asp111 in the essential Ca2+-dependent FVa subunit interaction. In the present study, FV E96A, D102A and D111A were purified and evaluated for function, subunit dissociation and metal ion binding. Chromogenic and clotting assays in the presence of procoagulant vesicles showed that each variant was inhibited (approximately 20-40%). D111A was further inhibited (>90%) after cleavage by thrombin. Comparable function was observed on activated platelets. D111A inhibition correlated to spontaneous subunit dissociation and severely impaired Ca2+ binding. The Cu2+ interaction was also inhibited, suggesting interdependent Ca2+ and Cu2+ binding to FV. The parental FV (FV-810; wild-type human FV missing residues 811-1491) used here is fully active without proteolysis because the B domain is truncated. Therefore, a FVa-like functional configuration exists for intact D111A independent of normal metal ion interactions. Unlike D111A, the thrombin-mediated FVa derived from E96A and D102A had only moderately enhanced subunit dissociation upon chelation and had normal metal ion binding. For FV-810-, E96A- and D102A-derived FVa, loss of function after chelation significantly preceded subunit dissociation. This study defines the highly conserved segment spanning Glu96-Asp111 in FV as multifunctional. Of the three amino acids evaluated, Asp111 is essential and probably functions through direct and indirect effects on Ca2+ and Cu2+ interactions. Glu96 and Asp102 individually influence FV/FVa by more subtle effects, possibly at the metal ion-dependent subunit interface.