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Topics of this survey are the indications and use of plasma-derived and recombinant coagulation factor concentrates. These substitution therapies need to be handled carefully by weighing up their effectiveness against the long and short term side effects in the individual patient.

Several biological medicines derived from human and animal plasmas can effectively improve haemostasis in individuals with inherited or acquired defects in haemostasis. Factor VIII and factor VIII/vWF and factor IX concentrates are used to treat haemophilia A, von Willebrand disease and hemophilia B respectively. Cryoprecipitates are used to treat hypofibrinogenemia and von Willebrand disease where desmopressin (DDAVP) is ineffective or when plasma-derived factor VIII/vWF concentrates are unavailable. Thrombin-containing topical haemostatic agents and fibrin sealants are used to control perioperative bleeding. Intravenous immunoglobulin has several uses, including management of patients with autoimmune thrombocytopenias and patients with acquired factor VIII deficiency. Similar to most protein-based biological medicines, all the above products can elicit some level of antibody response, with clinical consequences that vary from mild anaphylaxis to loss of product efficacy. An ongoing potential safety concern with any biological medicine derived from blood/plasma is transmission of blood-borne pathogens. This safety concern has lessened significantly in the past decade as a result of the institution of more effective pre- and post-donation screening that tests for potential pathogens, and institution of pathogen reduction strategies to which many plasma-derived biological medicines are now routinely subjected. This article considers the manufacture, standardization, clinical efficacy and adverse event profiles of the plasma-derived biological medicines currently used to promote haemostasis in patients with inherited or acquired functional defects in haemostasis. It also considers approaches employed to minimize infectivity of biological medicines derived from human and animal plasmas and to manage patients who develop antibodies (inhibitors) to clotting factor concentrate infusions.

Developing countries provide clotting factor replacement for hemophilia patients using one or more of the following strategies. (1) Production of domestic plasma and cryoprecipitate provides some self-sufficiency but depends heavily on the presence of an excellent blood transfusion service. The risk of transmitting donor infections is not completely obviated even with good serologic testing. (2) Domestic plasma fractionation has been achieved in a handful of emerging countries but can be expensive. Patients may distrust businessmen of their own country and may be dissatisfied with the style of product available from domestic plants. (3) Contract fractionation has been successful when the donor country is able to provide adequate amounts of well-tested plasma. Its cost is not always lower than that of imported concentrate but a degree of national self-sufficiency is retained. (4) Importation of concentrate allows a wide choice of products. The selection should focus not only on price but also on product safety, which depends upon well-validated donor testing and viral inactivation. High levels of purification are not of great importance. Successful national hemophilia programs also address organization of care, sensible dosing, and equitable distribution of resources to gain the maximum benefit from scarce resources.

Treatment of patients with bleeding disorders (especially those with hemophilia) with blood products has been associated with infections with blood-borne viruses. Of these, hepatitis B and C viruses (HBV and HCV, respectively) and the human immunodeficiency virus (HIV) have created major health problems. Although virus-inactivation procedures have virtually eliminated these viruses from newer factor concentrates since 1985, the risk remains in developing countries where there is no ready access to these concentrates. Although a few of these countries have established their own fractionation facilities and in others the respective governments make concentrates available, the large majority of countries still face the problems of blood-borne infections. HCV will invariably lead to liver damage and many hemophiliacs who were exposed to the HCV virus will succumb to cirrhosis. Only approximately 10% of hemophilic patients infected with HCV will clear the infection naturally. Coinfection with HIV shortens the life expectancy. The HIV epidemic in hemophiliacs began in the mid-1980s. Patients in developed countries were especially affected because they were predominantly treated with factor concentrates that were manufactured from thousands of blood donors. Hemophiliacs in developing countries have considerably less HIV infection, although it does exist and depends largely on the source of the plasma fractions. Progress has been made not only in the purification of factor concentrates, but also in the understanding of the HIV virus and in the development of antiretroviral treatment modalities. However, there are still several challenges in delivering antiretroviral treatment that must be addressed before the full impact of these transmitted infections is known.

The abilities of three synthetic oligosaccharides to accelerate antithrombin inhibition of ten clotting or fibrinolytic proteinases were compared with those of unfractionated, fractionated high-affinity and low-molecular-weight heparins. The results show that the anticoagulant effects of the latter three heparins under conditions approximating physiologic are exerted almost exclusively by acceleration of the inactivation of thrombin, factor Xa and factor IXa to near diffusion-controlled rate constants of approximately 10(6) - 10(7) M(-1).s(-1). All other proteinases are inhibited with at least 20-fold lower rate constants. The anti-coagulant ability of the synthetic regular (fondaparinux) and high-affinity (idraparinux) pentasaccharides is due to a common mechanism, involving acceleration of only factor Xa inhibition to rate constants of approximately 10(6) M(-1).s(-1) . A synthetic hexadecasaccharide, containing both the pentasaccharide sequence and a proteinase binding site, exerts its anticoagulant effect by accelerating antithrombin inactivation of both thrombin and factor Xa to rate constants of approximately 10(6) - 10(7) M(-1).s(-1), although thrombin appears to be the more important target. In contrast, factor IXa inhibition is appreciably less stimulated. The conformational change of antithrombin induced both by the pentasaccharides and longer heparins contributes substantially, approximately 150-500-fold, to accelerating the inactivation of factors Xa, IXa and VIIa and moderately, approximately 50-fold, to that of factor XIIa and tissue plasminogen activator inhibition. The bridging effect due to binding of antithrombin and proteinase to the same, long heparin chain is dominating, approximately 1000-3000-fold, for thrombin inhibition and is appreciably smaller, although up to approximately 250-350-fold, for the inactivation of factors IXa and XIa. These results establish the proteinase targets of heparin derivatives currently used in or considered for thrombosis therapy and give new insights into the mechanism of heparin acceleration of antithrombin inhibition of proteinases.

Immunological abnormalities have been reported in haemophiliacs. Although infections with HIV, hepatitis and other viruses may contribute to these abnormalities, immune defects are detectable also in HIV seronegative haemophiliacs. It is likely that chronic exposure to extraneous proteins in clotting factor concentrates (CFCs) may play a role in immunomodulation, but the underlying mechanisms remain unclear. The results of the present paper show that: a) soluble HLA class I (sHLA-I), soluble Fas-ligand (sFas-L) and transforming growth factor beta 1 (TGF-beta1) are detectable in plasma derived but not in recombinant CFCs; b) the level of sHLA-I and sFas-L is proportional to the grade of CFCs purity whereas TGF-beta1 showed very variable levels; c) soluble molecules detected in CFCs exert immunomodulatory effects in vitro like apoptosis induction in Jurkat cells and inhibition of mixed lymphocyte reaction response, antigen-specific lymphocyte cytotoxic activity and neutrophil chemotaxis.

Three commercial prothrombin complex concentrates (PCC) were compared in vitro. Differences in the activities or contents, respectively, of the PCC factors FII, FVII, FIX, FX and the proteins C, S, and Z (antigen) in particular were found. Global tests of activated factors did not reveal marked differences between the products. Neither free thrombin nor elevated levels of activated FX were detected. In contrast, analyses of FVIIa, of residual amidolytic activities, and of concentrations of unwanted ingredients demonstrated considerable differences between the products. While antithrombin III was detected only in two of three concentrates, heparin was found in all PCCs but in markedly different concentrations. Although the homogeneity of the products has been clearly improved in comparison with former comparative investigations, the products can be distinguished by their compound profile and by a couple of in vitro assays.

Plasma fractionation has emerged as one of the most scientifically demanding fields in the biopharmaceutical area. Producing safe plasma derivatives implies the development, use, and proper understanding of sensitive testing technologies to detect infection markers in starting plasma. It also requires the implementation of carefully selected, nondenaturing, efficient plasma protein purification and viral reduction technologies that do not alter the physiological functions and clinical potential of plasma proteins. Success in this field can be achieved only by a strong commitment to sustain constant research and development of projects targeting the production of safer and innovative plasma products.

It is known that parvovirus B19 (B19) is transmitted to hemophiliacs by clotting factors prepared from human plasma. However, it is not clear whether B19 is also transmitted by the more recently used inactivated clotting factor preparations. Therefore, we investigated 69 hemophiliacs, mostly children, receiving only virus-inactivated clotting factors. 49 of them (71%) were B19 IgG-positive and 18 of the IgG-positive hemophiliacs (37%) were also B19 IgM-positive. In contrast, out of 73 age-matched controls only 10 (14%) were IgG-positive, two of them being also IgM-positive. In hemophiliacs treated before 1984 with non-inactivated clotting factors, seroprevalence was very similar: 94/136 (69%) presented B19 IgG antibodies as compared to their age-matched controls with 16/50 (32%). Out of the 94 IgG-positive patients 24 (26%) were IgM-positive, whereas IgM antibodies were never found in 16 sera of 16 IgG-positive controls. In 4 out of 24 IgM positive hemophiliacs, B19 DNA was detected in the sera by using the polymerase chain reaction. However, B19 DNA was also found in 3/69 anti-B19 IgM-negative, HIV-infected hemophiliacs (all three patients in CDC stage IV). Since it seems unlikely that the results only represent passive acquisition of B19 DNA from blood products and induction of antibodies by immunization with inactivated antigen, the observations rather suggest that infection with B19 is transmitted by clotting factors, including those treated for virus inactivation.

Phospholipid procoagulant material mainly derived from platelets interferes or "bypasses" the more specific tests for lupus anticoagulants. Such material in test plasmas can be inactivated with recovery of lupus anticoagulant activity by simple extraction with chloroform. This solvent treatment damages mainly factors VIII, V, VII and IX. Ether and various other solvents were less damaging to these clotting factors but not quite as effective as chloroform in the recovery of lupus anticoagulant activity.

We studied activation of human coagulation factors IX and X by factor VIIa in the presence of calcium ions, phospholipid (phosphatidylserine/phosphatidylcholine, 50/50, mol/mol) and purified tissue factor apoprotein. Activation of factor IX and factor X was found to occur without a measurable lag-phase and hence initial rates of factor IXa and factor Xa formation could be determined. Like previously observed for the activation of factor X, the activation of factor IX was saturable with respect to factor VIIa, tissue factor apoprotein and phospholipid. The results suggested that in the presence of a Ca2+ ions the same ternary complex of factor VIIa-tissue factor apoprotein-phospholipid is responsible for the activation of factor IX and factor X. Both the apparent Km of 22 nM-factor IX and the apparent Kcat of 28 min-1 were about 3-fold lower than the corresponding parameters of factor X activation by this complex. Hence, the catalytic efficiency (Kcat/Km) of factor IX and factor X activation was about equal. However, the two substrates inhibited the activation of each other by competition for the same catalytic sites. The apparent Kinh of factor IX for inhibition of extrinsic factor X activation is 30 nM. The apparent Kinh of factor X for inhibition of extrinsic factor IX activation is 116 nM. From these kinetic data it was calculated that at plasma concentration of factors IX and X, the rate of extrinsic factor IX activation would be half the rate of factor X activation.(ABSTRACT TRUNCATED AT 250 WORDS)

The behaviour of contact factors, complement components and antiproteases during the preparation of prothrombin complex concentrates by adsorption of the clotting components on DEAE-Sephadex has been studied. The pro-enzymes: factors XII, XI and prekallikrein were removed by pre-elution in function of the salt concentration. In contrast, high molecular weight kininogen was considerably enriched in PCC preparations. C4 of the complement system displayed an analogous behaviour. C1s reached a 4-5 fold plasma concentration but C3 only 30% of the normal plasma level. The prothrombin complex concentrate contained no antithrombin III nor alpha 2M nor alpha 2 antiplasmin but a three fold plasma concentration of C1-inactivator and a 15 fold increase of inter-alpha-trypsin inhibitor. NAPTT (Non Activated Partial Thromboplastin Time) ratios did not seem to be in accordance with either the presence or the absence of contact enzymes. Moreover 0.20 M NaCl appeared as the minimal pre-elution molarity necessary to ensure a NAPTT ratio above thrombogenic values. Molecular alteration of high molecular weight kininogen and C4 was observed and its significance discussed. Complex formation between C1-inactivator and proteases was shown to be another sign of undesirable proteolytic events.

An anticoagulant activity was isolated from the plasma of a patient with a strong lupus-like anticoagulant using gel filtration by high performance liquid chromatography. IgM were detected in this anticoagulant fraction which exhibited specificity towards 50% phosphatidylcholine - 50% phosphatidylserine vesicles and cardiolipin. These phospholipids were able to produce an apparent 3-fold enhancement of purified human protein C activation by human alpha-thrombin in the presence of purified human placenta thrombomodulin. In the absence of phospholipid, the anticoagulant fraction had no effect on thrombomodulin activity. The anticoagulant fraction could neutralize the enhancement of thrombomodulin activity by phospholipid in a dose-dependent manner. This study suggests that the neutralization of phospholipid might result in a reduced activation of protein C which could be responsible for the occurrence of thrombotic complications in a proportion of patients with lupus anticoagulants.

The hemostatic abnormality in 18 patients with adult T cell leukemia (ATL) was studied. Activated partial thromboplastin time (APTT) was slightly prolonged and prekallikrein activity was markedly low in these patients. The leukemic cell homogenate from these patients prolonged the recalcification time (RCT) of normal plasma; homogenates containing more than 3 x 10(3) cells/microliter prolonged it, although a lower cell concentration shortened it. The crude anticoagulant fraction from the gel filtration, with a molecular weight of about 34,000, prolonged RCT. The crude anticoagulant did not affect prothrombin time (PT), thrombin activity or activated X activity at any concentration, but prolonged the contact activation test, inhibited the activation of prekallikrein and prolonged RCT of Fletcher trait, Fitzgerald trait and F XII deficient plasma. These effects of ATL cell homogenate were stronger on platelet poor plasma than on platelet rich plasma. Although ATL cells had low procoagulant activity, increase of leukemic cells made anticoagulant activity predominant, might be the cause of hemostatic abnormality or amplify the bleeding tendency in patients with ATL.

Clotting enzyme activity was detected in lysates prepared from the coelomocytes of Asterias forbesi following incubation with endotoxin-activated Limulus amebocyte lysate. This activity was not detected in the cell-free coelomic fluid. The enzymatic activity from the sea star lysate hydrolyzed the synthetic substrate S2222 but not S2238 or S2251, and polymerized partially purified Limulus clottable protein. The clotting enzyme activity was not detected following treatment of the sea star cell lysate with endotoxin or with the clotting enzyme from Limulus lysate. The enzymatic activity, generated in sea star cell lysate by the activated Limulus lysate, was inhibited by the addition of benzamidine; and its effect was suppressed with rabbit anti-sera directed against Asterias whole cell lysate, but not with anti-sera directed against the previously reported Sea Star Factor.