Oscar D. Ratnoff

Morphologic characteristics of adsorbed human plasma proteins on vascular grafts and biomaterials.

Protein adsorption on the surfaces of clinically significant prosthetic vascular graft materials from human whole blood was independent of plasma concentration as determined morphologically by use of immunogold labels. Some proteins, such as fibrinogen, adsorbed in a multilayer pattern on expanded polytetrafluoroethylene and had a preference for particular surface features of the polymer. Other proteins, such as Hageman factor (factor XII), showed diffuse adsorption patterns. Physiologically significant proteins that have not been well studied, such as immunoglobulin G and factor VIII, adsorbed readily to the surface of expanded polytetrafluoroethylene. This finding may be significant since adsorbed proteins may activate coagulation mechanisms and immunologic responses, including platelet and monocyte adhesion and activation. Any human blood protein for which an antibody has been developed can be studied by use of this technique.

Hageman Factor: Alterations in Physical Properties during Activation

Highly purified preparations of Hageman factor, a potent clotpromoting agent in normal mammalian plasma, had a sedimentation coefficient of approximately 5S before activation. After activation, the Hageman factor behaved as a much less soluble or larger molecule during ultracentrifugation and gel filtration. No significant change in sedimentation behavior was noted when the Hageman factor in plasma deficient in plasma thromboplastin antecedent was activated. The altered sedimentation behavior of purified activated Hageman factor probably reflects its decreased solubility in aqueous media.

Inhibition of ellagic acid-activated Hageman factor (factor XII) and Hageman factor fragments by popcorn inhibitor.

Abstract An inhibitor derived from popcorn prolonged the partial thromboplastin time of normal human plasma. The effect of this agent was localized to its inhibition of activated Hageman factor (factor XII), as demonstrated in amidolytic assays employing ellagic acid-activated Hageman factor and the carboxy-terminal fragment of Hageman factor (HFf.

FIBRIN CROSS‐LINKING and HEREDITY*

Several years ago, we undertook to review the literature concerning the inheritance of fibrin-stabilizing factor (Factor XIII) deficiency. In the kindred originally studied by Duckert,? four males and four females were affected. Strikingly, recurrent consanguinity was present in this kindred. These characteristics suggested that the inheritance of fibrin-stabilizing factor deficiency had the characteristics of an autosomal recessive trait. This view was challenged by H a m p t ~ n , ~ who believed that in some families the disorder might be inherited in an X chromosome-linked recessive manner. We carefully examined the family trees of the cases which had been reported through the year 1967, and filled in details missing from the original publications by correspondence with the patients physicians. We found, to our surprise, that the patients seemed to fall into two groups: in 12 families, only males were affected, while in 9 others, females were said to have the disease. Curiously, among the families in which only males were affected, consanguinity was present in only 1 out of 12 instances. In contrast, among the families in which female patients were known, the patients were the offspring of consanguineous unions in 5 out of 9 instances. The possibility that this bizarre distribution had occurred by chance was about 1 in 33. The possibility that the inheritance of fibrin-stabilizing factor deficiency might occur in two different ways was challenged by Lorand and his associates in 1970, leading us to reexamine the data.& Again, we were led to the same conclusion. Now, we have again summarized the information available in all families known to us, both those published in the literature and those which we have learned about from colleagues in the field. TABLE 1 summarizes what we know about the inheritance of fibrin-stabilizing factor deficiency in these families. We are familiar with some 56 families in which the diagnosis seems well established. In 24 of the families, only males were afflicted with the disease, while in 32 other families, females were affected, Among the 24 families in which only males had fibrin-stabilizing factor deficiency, consanguinity was recognized in only 4 instances. Two of these four families were studied in India, where consanguinity is especially frequent. In contrast, among the 32 families in which there were affected females, consanguinity occurred in 19 families. The possibility that this distribution could have occurred by chance is only 1 in 200. Presumably, then, whatever the explanation, there are indeed two different groups of patients with fibrin-stabilizing factor deficiency. In one

Effect of Some Analogues of Bradykinin Upon Vascular Permeability.

Summary The decapeptide, lysyl brady-kinin (Kallidin I) was more effective in increasing vascular permeability in the skin of guinea pigs than was the nonapeptide, bradykinin, or Kallidin II. Bradykinin has been reported to be more active in some other assays measuring pharmacological effects than lysyl bradykinin. All other analogues of bradykinin tested were less active than bradykinin in the permeability assay with the exception of glycyl bradykinin, another decapeptide, which had an equal effect. While an inactive analogue of bradykinin did not block increased vascular permeability induced by a later injection of bradykinin, two active compounds, lysyl bradykinin and bradykinin inhibited the effect of a subsequent injection of either one into the same site. Synthetic bradykinin BRS-640 was generously provided by Dr. Harry Baird, Sandoz Pharmaceuticals, Hanover, N. J. Dr. Ernest D. Nicolaides generously provided bradykinin analogues, which were synthesized in the Research Laboratories of Parke Davis & Co., Ann Arbor, Mich. Dr. Miklos Bodanszky Dept. of Chemistry, Western Reserve University, has provided helpful criticisms of this manuscript.

Studies on a circulating anticoagulant inhibiting factor XI in a patient with congenital deficiency and carcinoma of the prostate

An inhibitor of plasma thromboplastin antecedent (PTA, factor XI), measured in coagulant and radioimmunoassays, was detected in a 60‐year‐old man with carcinoma of the prostate who had no evidence of a bleeding tendency. Family studies indicated that the patient was either a homozygote or a heterozygote for hereditary factor XI deficiency. In contrast to earlier described patients with factor XI deficiency in whom inhibitors were detected, the patient was unaware of having been transfused with blood or blood products at any time before the discovery of the inhibitor. The inhibitor of factor XI in the patients plasma appeared to be predominantly in the IgG4 fraction and to be directed at a locus on the factor XI molecule other than the active site; it did not block the amidolytic properties of activated factor XI (XIa). Rather, it appeared to block adsorption of factor XI to negatively charged surfaces. The inhibitor interfered with measurement of other components of the intrinsic pathway of thrombin formation, perhaps explaining the low titres of other coagulation factors of the intrinsic system reported in patients with strong inhibitors directed against factor XL.

Brief Reviews: Some Recent Advances in the Study of Hemostasis

• High on the list of pathologic processes which impede the circulation with serious and often lethal consequences are thrombosis and its sequelae. Although the pathogenesis of thrombosis can vary in different situations, a major component of the usual thrombus is fibrin, the protein meshwork of blood clots. This review focuses on some of the newer information concerning the clotting process and its inhibition, the relationship between clotting and such defense mechanisms as generation of kinins and fibrinolysis, and the role of complement in blood coagulation. It attempts to present a sample of present trends rather than a compendium of newer knowledge.

Current Clotting Concepts in Urology

A modern outline of the physiology of coagulation, a simple approach to the use of laboratory investigation for patients with possible clotting disorders in unexplained hematuria and several illustrative cases are presented. The diagnosis and management of localized fibrinolysis in the genitourinary tract and intravascular coagulation as possible causes of post-prostatectomy hemorrhage are discussed. A minimum of a platelet count, fibrinogen assay and, if possible, measurement of fibrinogen-related antigens should be obtained before administration of epsilon aminocaproic acid.

Confirmation of Mendelian Properties of Heterodimeric Fibrinogen Molecules in a Heterozygotic Dysfibrinogenemia, “Fibrinogen Amarillo,” Using GPRphoresis to Differentiate SemiFibrin Molecules from Fibrinogen and Fibrin

The fibrinogen molecule consists of two sets of Aalpha, Bbeta, and gamma chains assembled into a bilateral disulfide linked (Aalpha, Bbeta, gamma)2 structure. Cleavage of the two A-fibrinopeptides (FPA, Aalpha1-16) from normal Aalpha chains with arginine at position 16 (RFPA) by thrombin or the venom enzyme atroxin transforms fibrinogen into self-aggregating fibrin monomers (alpha, Bbeta, gamma)(2). Mutant Aalpha16R-->H fibrinopeptide (HFPA) cannot be cleaved from fibrinogen by atroxin. Many studies on heterozygous dysfibrinogenemias with this mutation suggested that incorporation of the mutant chains into the molecules was ordered in a manner yielding only (1) homodimeric normal (RFPARFPA) atroxin-coagulable molecules and (2) homodimeric abnormal (H(FPA)HFPA) atroxin-incoagulable molecules in equal quantities. Although heterodimeric molecules (RFPAHFPA) could not be found in studies on the intact protein, Meh et al. demonstrated their existence by showing that CNBr digests of fibrinogens from atroxin-treated Aalpha16R-->H heterozygotic dysfibrinogenemias consistently yielded N-terminal fragments (NDSKs) with partially resolved electrophoretic bands predominantly in between the NDSKs of fibrinogen and alpha-fibrin. An opportunity to confirm and better quantify the heterodimers arose with the recent development of a method (GPRphoresis) for identifying molecules lacking only one FPA, which is applied here in study of a newly presenting case of an Aalpha16R-->H dysfibrinogenemia, fibrinogen Amarillo. GPRphoresis uses electrophoretic shifts, staged with GPRP-NH(2) to separate the self-aggregating fibrin monomers lacking both FPAs from weakly aggregating semifibrin molecules lacking one FPA An antifibrin alpha17-23 antibody is used to measure and differentiate the semifibrin from fibrinogen with FPA fully intact. Applying GPRphoresis to atroxin digests of fibrinogen Amarillo clearly demonstrated RFPARFPA, RFPAHFPA, and HFPAHFPA molecules in nearly perfect Mendelian 1:2:1 proportions. In turn, the high levels of the semifibrin in the terminal atroxin digests provide genetic phenotypic evidence supporting fidelity of the GPRphoresis method.

Some complications of the therapy of hemorrhagic disorders

The principal mode for treating disorders of hemostasis is correction of the patients functional defect by transfusions of appropriate fractions of normal plasma or transfusions of platelets. Two major complications of such therapy are the transmission of infectious diseases, particularly hepatitis and the acquired immune deficiency syndrome (AIDS), and the development of antibodies against clotting factors that are deficient in the patients plasma. Measures that reduce the occurrence of infection include careful selection of donors, fractionation of plasma with the help of monoclonal antibodies, and treatment of plasma or its fractions with heat or with virus-inactivating organic solvents. No technique of preparing or administering blood or its components can prevent the emergence of antibodies against clotting factors. Desensitization by repeated infusions of antigen, for example, antihemophilic factor, however, appears to result in remission in some patients.