Measurement of fibrinolytic components in human tissue

Abstract

The fibrinolytic system is involved in the resolution of thrombi and in tissue repair. Quantitation of the activators and inhibitors of this system at tissue level is crucial to further characterise these processes. Hitherto, there have been difficulties in measuring the individual components of the plasmin system in human vascular and peritoneal tissue. The aim of this study was to develop a protocol allowing quantitation of activators and inhibitors of plasmin generation at the tissue level. Following a strict protocol in the processing of tissue, the efficiency of extraction in tissue homogenisates was compared using buffers containing acetic acid, 1% Triton X-100 and thiocyanate. The influence of different modalities of normalisation was investigated by normalising to wet weight, total protein content, protein content in supernatant or DNA. Using the protocol, all buffers extracted components of the plasmin system sufficiently for detection. The acetic acid buffer yielded the greatest amount of protein, and in extracting plasminogen activators was comparable to the thiocyanate buffer and significantly more efficient than the Triton buffer (p<0.05). The relationship between the individual components was unaltered by different means of normalisation. The protocol described, using an acetic acid buffer and normalising to wet weight, seems to be a simple and efficient technique for measuring components of the fibrinolytic system, at least in the tissues investigated.

Key Words:

• PAI
• peritoneum
• tissue
• t-PA
• uPA
• vessels