Hemorrhagic Dysfibrinogenemia Undetectable By Clinical Coagulation Tests and Identified By Isolated Fibrinogen Investigations (Crypto-Dysfibrinogenemia)

A pilot study of isolated 7μM fibrinogen (fg) from 17 probands, with typical clinical coagulation test results and different (heterozygous) missense mutations, yielded acceleration of visually determined clot lysis (CL), which was induced by recombinant tissue plasminogen activator (rtPA,190 nM) in15% afibrinogenemic plasma, pH 7.4. Mean CL time was 44 minutes, control 190 minutes. The diagnosis in a few probands was suspected by mildly abnormal clinical laboratory test results suggesting that normal clinical test results can mask the presence of dysfibrinogenemia. To test this hypothesis, we investigated the isolated fg of three probands with chronic hemorrhagic diathesis, unexplained by other underlying disease(s), non-diagnostic clinical coagulation test results, and a similarly hemorrhagic history in at least one other family member. Apart from each proband's more specific history, typical presentation included chronic unprovoked bruising, prolonged wound bleeding, ecchymoses, and/or hematomas developing within ∼24 post-operative hours. More specifically, fg Jamaica was from a 69 year old woman with a history of invariable menorrhagia, three first trimester miscarriages, and excessive bleeding following tonsillectomy (requiring transfusion), removal of a deceased fetus, each of two full term deliveries, hysterectomy (requiring transfusion), and laminectomy resulting in a massive hematoma requiring surgical evacuation and transfusion. Fg Forest Hills was from a 55 year old woman who developed excessive bleeding from tonsillectomy requiring re-packing, hysterectomy requiring re-exploration, bilateral mastectomy and plastic repair, oopharectomy, urinary bladder polypectomy, and dilatation and curretage requiring procedure repeat and cauterization. Fg Port Jefferson was from an 82 year old woman who developed excessive post-operative wound bleeding following urinary bladder-rectal fistula repair, and from a knee operation requiring transfusion. Recently she underwent radical mastectomy for malignancy, and owing to our results her physician placed her on a standard epsilon amino caproic acid regimen extending to five post-operative days. There was no post-operative bleeding but wound seromas developed requiring drainage. For our investigations, thromboelastography (TEG) was used to measure clot strength (stiffness) and CL. Clots of 7 μM fg (>95% clottable) were induced by 0.5 U/ml thrombin in Tris-HCl buffer, µ=0.15, pH 7.4, containing 10 mM CaCl2, rtPA, and afibrinogenemic plasma (vide supra). For clot turbidity 3 μM fg, pH 6.4, and thrombin 0.2 U/ml were used. SDS-PAGE analyses of cross-linked and non-cross-linked clots revealed no abnormalities. Using the Malvern Nano Rheometer, clots from 3 μM fg were examined at 1 Hz frequency with increasing oscillation magnitude plotted as a function of shear amplitude expressed in Pascal (Pa) units and reflecting G'(shear modulus) and G” (complex modulus). Proband clots, Table 1, displayed accelerated CL and decreased G', suggesting abnormally formed fiber networks with increased susceptibility to fibrinolysis. Owing to its lowest G', non-enzymatically induced networks of fg Port Jefferson were also examined by atomic force microscopy (AFM). These were induced by fg adsorption on polystyrene surface using our published procedure. Topographic and lateral AFM scans disclosed a strikingly dense network consisting of fine, almost uniform diameter fibers with pronounced branching, in sharp contrast to the variable diameter, long control fibers. Clearly, rheometer and CL measurements were more sensitive in detecting abnormal clots than their turbidity and TEG counterparts. Also, the three probands represent a distinct and novel dysfibrinogenemic population identifiable by isolated fg but not by clinical laboratory investigations. Apparent efficacy of anti-fibrinolytic therapy following diagnosis of one proband underscores the potential clinical relevance of the foregoing investigations.