Welcome: Neville Marsh, Chair

Francis S. Markland, R. Manjunatha Kini, Co-chairs

Introduction of new Registry member: Dr. Takashi Morita

Retirements: Hubert Pirkle, Che-Ming Teng, Fritz Markwardt. Letters of thanks will be sent to these retiring members.

Dr. Markland reported on recent work on fibrolase including coupling with the cyclic RGD peptide, Diatide P734. This chimeric compound retained azocasein and fibrin plate activity and K_m and V_max were unaltered by coupling. The chimera inhibits platelet aggregation (IC₅₀ of 105nM, compared with an IC₅₀ of 76nM for P734 alone). The chimera binds via GPIIb/IIIa fibrinogen receptors, whereas fibrolase has no such binding activity. Studies to find the binding site of P734 on fibrolase revealed that there were seven lysine sites available. Digestion with endopeptidase LysC gave eight peptides and Lys₁₈₄ was identified as the site of P734 attachment, distant from the active site. It is anticipated that the chimera will be targeted to fibrin by fibrolase binding activity and thus brings platelet aggregating inhibitory activity to the thrombus. In vivo tests are in progress.

Dr. Pinango was unable to be present to give her report.

Dr. Marsh reported on new factors and recent work following publication of the first inventory in 1994. Previously identified hemorrhagins that have been further studied include rhodostoxin (Calloselasma rhodostoma), LHF-I and LHF-II (Lachesis muta muta) and HR2b (Trimeresurus flavoviridis). Rhodostoxin has been completely sequenced and is the first 4-disulphided hemorrhagin described. LHF-I and II have been studied in detail, in particular, their action on insulin bond cleavage. HR2b has also been completely sequenced. New hemorrhagins include the following: AaHIV (Agkistrodon acutus), a 44kD protein; ACL toxin 1 (Agkistrodon c. laticinctus) which has been completely sequenced and also appears in the previous inventory of fibrinogenolytic factors; BHRa and BHRb (Bitis arietans), large MW proteins (68 and 75kD); catrocollastatin, a large 50kD protein of the MDC family binding to collagen and VRH-I (Vipera russelli) a small 22kD non-zinc dependent protein. It was agreed that the inventory be updated by Dr. Marsh and submitted for publication as an official communication of the Registry.

Dr. Kini reviewed the main classes of factors that inhibit and/or induce platelet aggregation. Inhibitors include enzymic factors (metalloproteinases, nucleotidases, phospholipases) and non-enzymic factors include antagonists of GPIIb/IIIa, vWF-GPIb, thrombin-thrombin receptor interaction. Metalloproteinases include fibrinogenases, kistomin which cleaves GPIb and jararrhagin which cleaves the b 1 subunit of a 2b 1 integrin. Recent work on GPIIb/IIIa antagonists has been triggered by trigramin-echistatin publications and the possibility of developing therapeutic agents. They have a common function from a diverse structure including echistatin (49 residues, 4 disulphide bridges), kistrin (68 residues, 6 bridges), mambin and decorsin, all of which have RGD loops. Inducers of platelet aggregation include the serine proteinases, including the "thrombin-like enzymes, C-type lectins and factors from the sea anemone (equinatoxin) and sea cucumber. It was agreed that the inventory will be published as an official communication of the Registry and if possible as a review in Thrombosis and Haemostasis.

Dr. Patrick Gaffney gave an update on textilinin, a plasmin inhibitor from the common brown snake (Pseudonaja textilis) venom. This is a loose, fast reversible inhibitor

(K_I, 10^-7) which only inhibits plasmin and trypsin. Textilinin has a 47% homology with aprotinin and reduces bleeding in a mouse tail vein bleeding model.

It was decided not to proceed with an inventory of bacterial agents, as the number of such compounds was small.

Dr. Morita reported on various classes of C-type lectins. These are mainly heterodimers and include habu IX/X-bp, botrocetin (vWF), echicetin (GPIb), rhodocytin (Ia/IIa) and alboaggregin-B (Ib). Dr. Morita described the 3D structure of flavocetin A. It was agreed that the inventory be published as an official communication of the Registry. Dr. Kini will liaise with Dr. Morita over omission of C-type lectins from the inventory of platelet factors.

Dr. Kini will contact Prof. Teng to determine if a manuscript has been prepared on plant factors.

It was agreed that members of the Registry would be involved in a satellite on Animal Hemostatic Factors. Dr. Kini will approach Dr. Cassian Bon in Paris for his help. The meeting will be organized by Drs. Kini, Markland and Marsh.

This meeting concludes Dr. Marsh’s term of chairmanship and he formally handed over the chair to Dr. Manjunatha Kini. Co-chairs for the next period will be Drs. Francis Markland and Neville Marsh.

The next meeting of the Registry will be held in Paris, 4-5 July 2001.

The meeting was adjourned at 3:50pm.