Biomed & Pharmacorher Q Elsevier, Paris
1996; 50: 397400
Hemostasis, thrombosis
Endothelial
Cell Influence
S. Handt', Martina R.R. Hantgan2, and lInstieure Gemmy
of Pathology, and *Wake Forest
on Fibrinolysis
Meth', L. Tietze', C. Mittermayer'
and vascular endothelium
DETERMINATION OF SECONDARY FROM HUMAN PERIPHERAL BLOOD.
W.G.
STRUCTURE
OF NORMAL
FIBRIN
Jerome’,
dachen Umvers~fy of Technology, "niversiry, "inscon-Salem, NC, USA
cells (EC) actively participate in many physiologic pl-OC~SSt?S like inflammation, coagulation and fibrinolysis. EC influence fibrinolysis in both directions, proand antiflbrinolytic, by secretion of tissue Plasminogen Activator (C-PA) and its antagonist Plasminogen Activator Inhibitor 1 IPAI-1). To understand the role of EC in the situation of a fresh occlusive thrombus, which is i.e. the reason for a myocardial infarction, we developed an in-vitro model with a glass tube, coated with a confluent monolayer of HWEC (human umbilical venous endothelial cellsl. In the presence of differently stimulated EC (TNF-a, TGF%, PMA and Forskolin) a fibrin clot was formed by to a mixture of fibrinogen and adding thrombin plasminogen in a HEPES buffered GSY'S salt solution. After intervals of 2 and 4 hours the clot was slowiy perfused with a buffer solution additionaliy containing t-PA to induce lysis. Thrombus growth and lysis were measured by laser light scattering and lysis times were determined. In parallel the clots were stained for PAIand T:;;;yTd by intermediate voltage electronmicroscopy
Papineschi*
Edoardo and Enm Be”edeui*
Be”cdc”i*,
Fmilia
Bramaxi”,
Federico
Endothelial
We found that cytokine-activated EC strongly delay induced lysis of a fibrin clot by secreting PAI-1, which is bound to the fibrin strands. Therefore EC seem to be a major reason far "time dependent thrombolytic resistance", a term used to describe the reduced sufficiency of thrombolytic therapy starting more than 6 hours after the onset of the symptoms of a myocardial infarction. Additionally we present data about the behaviour of EC from different VSSCUlSr beds and show EC morphology and behaviour under the fibrin clot. t-PA
active
‘llx secondary svuclure of human fibrin fmm normal donors and fmm bovine and willine plasma was rmdied by FT-IR spenroscopy and a quantitative analysis of ils secondary smmm was suggeswd. For this purpxe. a previously experimenled specnum dxo”voluti”” pr”cedure was applied 10 the analysis of co”fomlador-senshive Amkk bands “d infixed swclrum. This wxedwe was atolied 10 A”& I and III analysis of bovine and suilline fibrin. obtainedindustrially. and’& Amide 111 analysis of hum& fibrin clots. The analysis of both Amide I and 111 in rile first ca$e was useful in adu 10 test the reliability of the method We found bovine. suilline and human fibrin 10 contain about 30%. a-helix (Amide I and 111 mmpone”ls a, 1653 cm-* and 1312 and 1284 cm-‘, re@vely). 40% B-sheas (Amide I and III wmpo”e”rs at 1625 cm-’ and 17.31 an-‘, rexecdvelvl and 30% funs IAnG& I and 111 co”xwnemS at 1696. 1680. 1675 cm’ and 12i9 071-l; respectively). The good agreement of our qua”titative data obtained separately by Amide I and Amide III analysis and consistem with a previous fibrinogen (fmm cannwcial sources) study, leads us 1” believe that the amwnts of secondary mwnues fcund are accurate. Cnly one information about B shea contellf of fibrin was already reported in literawe. but it &s not exiti any complete analysis of the Mha secondary sb-ucnlres. Fibrin, being a” insoluble pmtein. bar never been sm~cwalIy aml}sed by CD or W m&o&, which require soluble proteins; on rhe aher hand, X-Ray analysis has never been pdmhxl owing 1” ihe nmaystalline na”ue of rhe fibre “awork lk increase of t3 sheets in fibrin with respect 1” the libri”“ge” molecule could be due 1” the farnation of inter-molecular B sheets during the famadon of rhe staggered overlapping iibdllar aray and Leir lateral asscxiatio”. As rhe increase in 5 sheers ccntem occurs at d-e expense of a-helical swucturcs, it cadd also be suggested Lhat dx portion which undergixs the ccnfamaticaal tratiticm involves the solvent-exposed ahelix present in the central fragment E, which is sensitive fo thmmbi” attack, and ahelical portions present in the D distal globular domain
kindly supplled&d
for very hlplul
discussions.