Screening for fibrin specific monoclonal antibodies: the development of a new procedure.

The acquisition of monoclonal antibodies specific for human fibrin has been impaired by the similarity in chemical composition between fibrinogen and fibrin and the conformational difference between immobilised and soluble fibrinogen. Five monoclonal antibodies (mabs) with a known affinity for fibrin have been subjected to screening procedures which involved the presentation of different forms of both fibrinogen and fibrin to the test mabs. It was observed by scanning electron microscopy that dried fibrin (denoted fibrin D), immobilised on the wells of PVC plates was morphologically similar to the fibrin found in human clots whereas PVC-immobilised fibrin monolayers (fibrin M) and a homogenised form of fibrin (fibrin FF) presented two very different morphological appearances. It was shown that lack of cross reactivity of a mab with an antigen (e.g. fibrinogen) was validly demonstrated only when both mab and antigen were present in the soluble state. These findings have allowed the generation of a screening procedure which involves the use of fibrin D on PVC plates in conjunction with whole human plasma incubated with the test antibody. This screening procedure has been validated using two mabs, one of which has an exclusive fibrin affinity while the other has a broad spectrum crossreactivity with both fibrinogen and fibrin. This procedure would ensure the acquisition of all the five fibrin-specific mabs used in this study while other less reliable screening procedures might not.

Heterogeneity of thromboxane A2 (TP-) receptors: evidence from antagonist but not agonist potency measurements.

1. Thromboxane A2 (TP-) receptors in human, rat and rabbit platelets and in smooth muscle of guinea-pig trachea, rat aorta and rabbit aorta have been characterized by measurement of the potencies of agonists and antagonists having considerable variations in chemical structure. 2. On each washed platelet system, eight prostanoids induced maximal irreversible aggregation (full agonists) and the potency ranking was EP 171 > STA2 > 9,11-azo PGH2 > 9,11-endoxy-10a-homo PGH2 > U-46619 (standard) > PGH2 = 16-p-fluorophenoxy-omega-tetranor PGF2 alpha > 16,16-dimethyl PGF2 alpha. Correlations between the three platelet preparations for both absolute and relative potencies were good. On human platelets, STA2, at concentrations above that required for maximum aggregation, exerted an inhibitory effect which was independent of its interaction with the TP-receptor. 3. Five prostanoids, EP 109, EP 167, EP 204, PTA2 and 16,20-methano PTA2, exhibited partial agonist activity on the platelet and smooth muscle preparations. There was good agreement between absolute potencies on the six preparations; on platelets potency was assessed from shape change measurements, since aggregation, when present, always showed a very shallow concentration-response relationship. The magnitude of the maximum response induced by each compound decreased in the order listed above, to the extent that 16,20-methano PTA2 could be treated as a pure antagonist. 4. With U-46619 as agonist, the pA2 values of seven antagonists were found to be very similar on human and rat platelets. The potency ranking was EP 169 > AH 23848 > EP 092 > ONO 11120 > EP 115 = 16,20-methano PTA2 > BM 13177. There was a similar trend on rabbit platelets but pA2 values were 1.0-1.5 log units smaller; the exception was BM 13177 which had similar affinities. The antagonism produced by EP 169 and AH 23848 was surmountable on rabbit platelets but not on human and rat platelets. 5. None of the antagonists was highly potent on the rabbit aorta (pA2 values < 7.5 by Schild analysis). Affinities on the guinea-pig trachea and the rat aorta were higher and in the same range as those obtained for human and rat platelets. However the correlations of pA2 values between any pair of smooth muscle preparations and between any pair of platelet/smooth muscle preparations were either weak or not significant(P > 0.05). 6. The excellent agreement for both full and partial agonist potencies between the six preparations provides no evidence for TP-receptor subtypes and further suggests that the agonist recognition sites of the TP-receptors could be very similar, if not identical, in nature. In contrast, the different antagonist affinities found in this and other published studies indicate heterogeneity of TP-receptors. However, classification into TP,-, TP2-receptors, etc. on the basis of the limited antagonist data available does not appear appropriate at this time.

Imaging of human thrombi in the rabbit jugular vein: I: Comparison of two fibrin-specific monoclonal antibodies.

The development of monoclonal antibodies with a specificity for cross-linked fibrin may have a potential role in the detection and of thrombi and thrombolytic therapy. In this study, two monoclonal antibodies with a specificity for fibrin have been examined. In vitro studies have shown NIBn 123 (which has a high affinity for X-oligomer) and DD-3B6 to bind to immobilised fibrin on PVC plates as well as plasma clots which were incubated in the presence of plasma. The Km values for NIBn 123 and DD-3B6 wre 1.0 x 10(10)/7.7 x 10(8) M and 2.6 x 10(8) M respectively. No significant binding to fibrinogen either immobilised or in solution was found. The binding of these antibodies to a human thrombus in the jugular vein of the rabbit was monitored over a 24 hour period. Preferential binding of each antibody reached a ratio of approximately 1.0 (jugular/heart) at 24 hours and an image was detected.

Generation and partial characterization of five monoclonal antibodies with high affinities for fibrin.

Five fibrin-specific monoclonal antibodies have been generated using three different immunogens and a unique clot/plasma screening procedure. Two of the mAbs are directed to an epitope(s) on the A alpha-chain of fibrinogen while two others seem to react with a distinct B beta-chain epitope(s). A third antibody seems to displace all the mAbs from fibrin and may be directed to a repeating structural domain in the fibrin. The Kd values obtained (approximately 10(-9)M) using dried fibrin clots suggest high avidity for fibrin. All the mAbs cross-react with the fibrin-like X-oligomer structure in solution but in the presence of fibrin this reaction seems irrelevant due to the low Kd of the mAb/fibrin reaction. These mAbs have been examined for uptake by fibrin in a laboratory circulation circuit and while the uptake was dependent on mAb concentration an increase was noted for most concentrations over the first 2 h of circulation in the presence of plasma.

Changes in some aspects of platelet function with improvement of glycaemic control over 6 months.

Numerous platelet abnormalities, particularly hyperaggregation, have been described in diabetic patients, and it has been suggested that these may contribute towards the pathogenesis of microvascular complications. In the present study, the changes that occur in ADP-induced aggregation, sensitivity to a stable prostacyclin analogue (Iloprost), aggregation-induced thromboxane B2 production and platelet cyclic AMP levels were investigated in 9 young insulin-dependent diabetics, in which the glycaemic control significantly improved in one group (n = 5; HbA1 from 11.9-9.0%) over a 6 month period. With improvement of glycaemic control there was no significant change in the concentration of ADP required to produce 50 percent of the maximum aggregation wave response. However, there was a significant increase in the responsiveness of platelets to Iloprost and increased platelet thromboxane B2 production. There was no significant difference between the basal platelet cAMP levels before or after exposure to Iloprost. This study suggests that with improved short-term glycaemic control, although there are changes in platelet function, there may be no alteration in the homeostatic balance.