Full Text HL-95-010

COAGULATION, PLATELETS AND THROMBOSIS IN SICKLE DISEASE
PATHOPHYSIOLOGY

NIH GUIDE, Volume 23, Number 36, October 14, 1994

RFA:  HL-95-010

National Heart, Lung, and Blood Institute

P.T. 34

Keywords: 
  Blood Diseases 
  Cardiovascular Diseases 
  Pathogenesis 


Letter of Intent Receipt Date:  December 20, 1994
Application Receipt Date:  January 24, 1995

PURPOSE

The Division of Blood Diseases and Resources (DBDR) of the National
Heart, Lung, and Blood Institute (NHLBI), National Institutes of
Health (NIH), invites research grant applications to support basic
research on the role of hemostasis and thrombosis in the
pathophysiology of sickle cell disease vasculopathy.

HEALTHY PEOPLE 2000

The Public Health Service (PHS) is committed to achieving the health
promotion and disease prevention objectives of "Healthy People 2000,"
a PHS-led national activity for setting priority areas.  This Request
for Applications (RFA), Coagulation, Platelets and Thrombosis in
Sickle Cell Disease Pathophysiology, is related to the priority areas
of clinical prevention services, chronic disabling conditions, and
maternal and infant health.  The goal of this program is basic
research leading to approaches that ameliorate the long term
debilitating effects of this disease.  Potential applicants may
obtain a copy of "Healthy People 2000" (Full Report:  Stock No.
017-001-00474-0) or "Healthy People 2000" (Summary Report:  Stock No.
017-001-00473-1) through the Superintendent of Documents, Government
Printing Office, Washington, DC 20402-9325 (telephone 202-782-3238).

ELIGIBILITY REQUIREMENTS

Applications may be submitted by domestic and foreign for-profit and
non-profit organizations, public and private, such as universities,
colleges, hospitals, laboratories, units of state or local
governments, and eligible agencies of the federal government.  Awards
in response to this RFA will be made to foreign institutions only for
research of very unusual merit, need, and promise, and in accordance
with PHS policy governing such awards.  Applications from minority
individuals and women are encouraged.  Foreign institutions are not
eligible for FIRST (R29) awards.

MECHANISM OF SUPPORT

This RFA will use the National Institutes of Health (NIH) individual
research project grant (R01) and FIRST award (R29) and is a one-time
solicitation.  Applicants, who will plan and execute their own
research programs, are requested to furnish their own estimates of
the time required to achieve the objectives of the proposed research
project.  Up to 46 months of support may be requested for R01
applications, but 58 months of support must be requested for R29
applications.  At the end of the official award period, renewal
applications may be submitted for peer review and competition for
support through the regular grant program of the NHLBI.  It is
anticipated that support for the present program will begin July 1,
1995.  Administrative adjustments in project period/or amount of
support may be required at the time of the award.  Since a variety of
approaches would represent valid responses to this RFA, it is
anticipated that there will be a range of costs among individual
grants awarded.  All current policies and requirements that govern
the research grant programs of the NIH will apply to grants awarded
in connection with this RFA.

FUNDS AVAILABLE

It is anticipated that for fiscal year 1995, $1,500,000 will be
available for this initiative.  It should be noted that award of
grants pursuant to this RFA is contingent upon receipt of such funds
for this purpose.  It is anticipated that about six new grants will
be awarded under this program.  The specific number to be funded
will, however, depend on the merit and scope of the applications
received and on the availability of funds.  If collaborative
arrangements involve sub-contracts with other institutions, the NHLBI
Grants Operations Branch (telephone: (301) 594-7436) should be
consulted regarding procedures to be followed.

RESEARCH OBJECTIVES

Vascular occlusive events account for much of the morbidity and
mortality associated with sickle cell disease.  Despite significant
advances in clinical observation and experimental investigation,
however, the pathogenesis of these vaso- occlusive events remains
ill-defined and the subject of much speculation.  Factors
contributory to the initiation of vaso-occlusion may include
rheological abnormalities related to sickling, abnormal adhesiveness
of sickle red cells to endothelium, dehydration and poor
deformability of sickle red cells, various vascular factors, and
activation of hemostatic systems.  Despite various observations that
are consistent with a pathogenetic role for hemostasis and thrombosis
in sickle vaso-occlusive events, the incompleteness of existing data
leaves important but unanswered hypotheses regarding this neglected
area of sickle disease pathophysiology.  Further understanding of the
role of thrombosis and/or hemostatic activation in sickle cell
disease pathophysiology is likely to facilitate the rational design
of therapeutic trials and approaches and to thereby improve clinical
care of affected individuals. Clinical Involvement

Cerebrovascular disease contributes significant morbidity in up to 17
percent of patients with sickle cell anemia, with stroke resulting
from thrombosis usually developing at sites of underlying intimal
hyperplasia.  Suggestive, albeit inconclusive, evidence suggests that
thrombosis may contribute to the acute chest syndrome and pulmonary
hypertension in sickle cell disease.  Whether this disease includes
an increased risk for venous thrombosis and thromboembolism has not
been resolved.  The possibility that thrombosis and/or hemostatic
activation contributes to other manifestations such as the acute
painful vaso-occlusive crisis has not been excluded.  Limited trials
have failed to find benefit in prophylactic aspirin, although this
approach could leave thrombin-induced platelet activation unimpeded.
Very small trials have claimed lack of benefit from warfarin therapy
but discernible benefit from long-term subcutaneous heparin.  There
have been no studies of antiplatelet or anticoagulant agents in the
primary or secondary prevention of stroke in sickle cell disease, nor
has the efficacy of thrombolytic agents been tested.

Pathophysiology

Insofar as thrombotic complications occur in sickle cell disease,
they may be uniquely widespread in their anatomical distribution,
with abnormal clotting occurring in the venous (e.g., thrombosis with
pulmonary embolus), arterial (e.g., stroke), and microvascular (e.g.,
painful vaso-occlusive crisis) circulations.  Such heterogeneity of
clinical phenomena is distinctly unusual (i.e., in inherited
hypercoagulable states such as protein S or C deficiency) and
conceivably could derive from participation of multiple hemostatic
disturbances.  Virchow's triad (altered blood flow, disturbed
vascular integrity, and alterations in the blood) encompasses our
understanding of the pathogenesis of thrombosis, irrespective of the
anatomical vascular bed in question.  Therefore, it is reasonable to
consider the potential role of hemostasis and thrombosis in sickle
cell disease in terms of the components of Virchow's triad.  Blood
flow augments mass transport of blood borne elements beyond that
resulting from diffusion alone and may thereby influence the
thrombotic process.  In a normal laminar flow situation, the
effective concentration of platelets is increased several fold
towards the periphery of a normal vessel.  This cellular distribution
is a function of both red cell concentration and wall shear rate.
Flow models have been employed to study a combination of normal red
blood cells and platelets, but nothing is known about sickle
erythrocytes and their interaction with platelets in such models.
The complex interplay of abnormalities in erythrocyte deformability,
membrane characteristics, vascular factors (e.g., flow rate, vessel
diameter and reactivity, and so on), and tendency to adhere to the
vascular endothelium has not been directly studied in the context of
the genesis of thrombosis.  The disturbance in normal laminar blood
flow in sickle vessels might directly influence endothelial cell
function; for example, it has been previously demonstrated that shear
stresses regulate the synthesis and expression of prostacyclin,
tissue plasminogen activator, and von Willerbrand factor by
endothelial cells. Studies have also shown that platelets exposed to
physiological shear forces ex vivo (in viscometers) aggregate without
the addition of exogenous agonists, a phenomenon not seen in standard
platelet aggregation assays.  Further studies specifically examining
shear stresses in interrelationships between sickle red cells,
platelets, endothelial cells, and coagulation factors may enhance our
understanding of this disease.

Normal vascular endothelium plays an important role in resisting
thrombosis.  It is likely that endothelial cell dysfunction (and
possibly denudation) contributes significantly to the thrombotic
manifestations of sickle cell disease.  Increased numbers of
circulating endothelial cells have been observed during crisis, and
elevated levels of prostacyclin metabolites have been variably
observed in sickle plasmas, possibly suggesting that endothelial
injury occurs in vivo.  Vascular intimal hyperplasia of large
cerebral arteries, sometimes with superimposed thrombosis, is thought
to be the most frequent cause of stroke in sickle cell disease.  It
is now possible with a combination of magnetic resonance imaging and
angiography to demonstrate the presence of vascular intimal lesions
in the cerebral vascular circulation of children with sickle cell
disease.  In order to extend our knowledge of the role of altered
vascular integrity in some of the other complications of sickle cell
disease, further histopathological and clinical-pathological data are
necessary to define the frequency and significance of intimal
hyperplasia in other vascular beds.  This in turn raises the question
of the pathogenesis of this vascular lesion and whether it may derive
from endothelial injury caused by adherent sickle erythrocytes or
molestation of the endothelium by pathologic or biologic modifiers
that impact upon its anticoagulant/procoagulant balance.  For
example, elevated levels of tumor necrosis factor, IL1 and  endotoxin
have been noted in sickle plasmas, although the temporal relationship
of such changes to clinical or vascular events is undefined.  The
state of endothelial activation or integrity, either in general or in
relationship to specific clinical events, is wholly undefined in
sickle cell disease.

Numerous clinical studies have documented that the coagulation system
is in a state of activation in "steady state" sickle disease as well
as during painful crisis.  In particular it seems clear that ongoing
thrombin generation and fibrinolysis occurs in these patients (e.g.,
as evidenced by increased prothrombin F 1.2 fragment, fibrinopeptide
A, thrombin/antithrombin complexes, and D-dimer), even in steady
state; whether this change is in relationship to acute vaso-occlusive
crisis is less clear.  D-dimer levels are reported to be commonly
elevated in sickle patients with leg ulcers, aseptic necrosis, or
stroke. Diminished levels of anti-thrombotic substances (proteins S
and C and antithrombin III) are often observed in sickle patients.
Since plasmatic coagulation is initiated by tissue factor in vivo, it
is significant that recent observation of accelerated Factor VII
turnover has provided indirect evidence for abnormal tissue factor
expression in sickle disease patients. Direct demonstration of tissue
factor expression, verification of its location (e.g., monocytes
versus endothelium), and identification of stimulants underlying its
apparent expression in sickle cell disease have not been attempted.
The extent to which hemostatic changes are contributed to in vivo by
abnormality of the sickle cell membrane (in which negatively charged
phospholipids are available to enhance coagulation reactions) or the
circulating red cell membrane spicular fragments in sickle plasma is
essentially unknown.

Other studies have provided suggestive evidence for platelet
activation (e.g., elevated plasma -thromboglobulin, decreased
platelet thrombospondin, and preliminary observation of abnormal
expression of platelet activation antigens) even in steady state
sickle patients, but unambiguous proof of this using modern, optimal
platelet collection methodology has not been provided.  The precise
reason for apparent platelet activation in these patients has not
been established, nor have the circumstances of its occurrence.  In
particular, the degree to which platelet abnormalities change
relative to specific clinical events is not defined, nor is the
relationship, if any, between platelet activation and thrombin
generation.  The recent demonstration that platelet-derived
thrombospondin could be a major factor in adhesion of sickle red
cells to endothelium identifies one potential link between the
hemostatic systems and vascular occlusion in this disease.  The
extent to which platelet release products impact upon the endothelium
and vessel wall in sickle disease has not been defined.

This RFA is intended to stimulate research on the role of hemostasis
and thrombosis in the pathophysiology of sickle cell disease.
Examples of research areas that would be responsive to this RFA are
given below.  This list is intended to provide guidance as to areas
suitable for study; it is not intended to be all inclusive.
Investigators are encouraged to consider other topics relevant to
this program.

o  Investigation of endothelial function/dysfunction as it pertains
to the anticoagulant/procoagulant balance of the endothelial surface
in sickle patients.  What is the state of endothelial activation in
sickle cell patients?  Does endothelial dysfunction contribute to the
coagulopathy and vasculopathy of sickle cell disease?  Do other known
factors peculiar to sickle disease (e.g., red cell adhesivity,
elevation of certain acute phase reactants) impact upon endothelial
cell hemostatic function?

o  Investigation of the reason(s) for coagulation abnormalities in
sickle cell disease.  What causes apparent activation of tissue
factor?  Where and under what circumstances is it expressed?  Does
the abnormal sickle red cell membrane or the presence of circulating
spicular fragments contribute to in vivo hemostatic abnormalities?
What explains apparent deficiencies of antithrombotic proteins in
these patients?

o  Investigation of the extent to which hemostatic abnormalities
contribute to sickle vascular disease.  What vascular events do (and
which do not) involve hemostatic abnormalities in their primary
pathogenesis?  For which events does hemostatic activation and/or
thrombosis play an important secondary role?  Does coagulopathy in
sickle disease precede or follow vasculopathy?

o  Investigation of platelet physiology in sickle cell disease.  What
is the extent of platelet activation in sickle patients?  Under what
circumstances does it occur, and what triggers it?  Is there any
relationship between platelet activation and specific vascular
events?

o  Investigation of the biochemical distinction (or similarity)
between acute painful crisis and inter-critical periods.  Does the
"steady-state" really exist vis a vis hemostatic systems? What are
the precise hemostatic differences between apparent steady state and
acute painful crisis?  Do any hemostatic abnormalities play a
different role in vaso- occlusive events other than the acute painful
episode?  Can detailed longitudinal studies of hemostatic systems
elucidate cause from effect, risk factor from primary trigger, acute
from chronic?

o  Can pharmacologic manipulation of hemostasis impact favorably upon
any vaso-occlusive manifestations of sickle cell disease?  The
primary intent of this RFA is to solicit basic rather than clinical
studies.  However, in recognition of the fact that the long term goal
of such work is to develop successful therapeutic approaches,
inclusion of some clinical investigation as a necessary and integral
part of a basic scientific program would be considered to be
responsive to this RFA.

SPECIAL REQUIREMENTS

Upon initiation of the program, the NHLBI will sponsor annual
meetings to encourage the exchange of information among investigators
who participate in this program.   Applicants should request
additional travel funds for one meeting each year to be held in
Bethesda, Maryland.  Applicants should also include a statement in
the applications indicating their willingness to participate in such
meetings.

INCLUSION OF WOMEN AND MINORITIES IN RESEARCH INVOLVING HUMAN
SUBJECTS

It is the policy of the NIH that women and members of minority groups
and their subpopulations must be included in all NIH supported
biomedical and behavioral research projects involving human subjects,
unless a clear and compelling rationale and justification is provided
that inclusion is inappropriate with respect to the health of the
subjects or the purpose of research.  This new policy results from
the NIH Revitalization Act of 1993 (Section 492B of Public Law
103-43) and supersedes and strengthens the previous policies
(Concerning the Inclusion of Women in Study Populations, and
Concerning the Inclusion of Minorities in Study Populations), which
have been in effect since 1990.  The new policy contains some new
provisions that are substantially different from the 1990 policies.
All investigators proposing research involving human subjects should
read the "NIH Guidelines for Inclusion of Women and Minorities as
Subjects in Clinical Research", which have been published in the
Federal Register of March 28, 1994 (FR 59 14508-14513), and reprinted
in the NIH GUIDE FOR GRANTS AND CONTRACTS of March 18, 1994, Volume
23, Number 11.

Investigators also may obtain copies from the program staff listed
under INQUIRIES. Program staff may also provide additional relevant
information concerning the policy.

LETTER OF INTENT

Prospective applicants are asked to submit, by December 20, 1994, a
letter of intent that includes a descriptive title of the proposed
research, the name, address, and telephone number of the Principal
Investigator, the identities of other key personnel and participating
institutions, and the number and title of the RFA in response to
which the application may be submitted.  Although a letter of intent
is not required, is not binding, and does not enter into the review
of a subsequent application, the information that it contains allows
IC staff to estimate the potential review workload and avoid conflict
of interest in the review.

The letter of intent is to be sent to:

Chief, Review Branch
Division of Extramural Affairs
National Heart, Lung, and Blood Institute
Westwood Building, Room 553
Bethesda, MD  20892
Telephone:  (301) 594-7478
FAX:  (301) 594-7407
Internet: James_Scheirer@NIH.GOV

APPLICATION PROCEDURES

Applications are to be submitted on the research grant application
form PHS 398 (rev. 9/91).  This form is available in an applicant
institution's office of sponsored research and from the Office of
Grants Information, Division of Research Grants, National Institutes
of Health, 5333 Westbard Avenue, Room 449, Bethesda, MD 20892,
telephone (301) 435-0714.  Use the conventional format for research
grant applications and ensure that the points identified in the
section on REVIEW CONSIDERATIONS are fulfilled.  FIRST applications
must include at least three sealed letters of reference attached to
the face page of the original application.  FIRST applications
submitted without the required number of references will be
considered incomplete and will be returned without review.

Applicants from institutions that have a General Clinical Research
Center (GCRC) funded by the NIH National Center for Research
Resources may wish to identify the GCRC as a resource for conducting
the proposed research. If so, a letter of agreement from either the
GCRC program director or principal investigator could be included
with the application.

To identify the application as a response to this RFA, check "YES" on
Item 2a of page 1 of the application and enter the title and RFA
number:  COAGULATION IN SICKLE CELL DISEASE  RFA HL-95-010.

The RFA label available in the PHS 398 (rev. 9/91) application form
must be affixed to the bottom of the face page of the application.
Failure to use this label could result in delayed processing of the
application such that it may not reach the review committee in time
for review.  In addition, the RFA title and number must be typed on
line 2a of the face page of the application form and the YES box must
be marked.

Send or deliver the completed application and three signed, exact
photocopies of it to the following, making sure that the original
application with the RFA label attached is on top:

Division of Research Grants
National Institutes of Health
Westwood Building, Room 240
Bethesda, MD  20892**

Send an additional two copies of the application to the Chief,
Centers and Special Projects Review at the address listed under
LETTER OF INTENT.  It is important to send these two copies at the
same time as the original and three copies are sent to the Division
of Research Grants.  Otherwise the NHLBI cannot guarantee that the
application will be reviewed in competition for this RFA.

Applications must be received by January 24, 1995.  If an application
is received after that date, it will be returned to the applicant
without review.  The Division of Research Grants (DRG) will not
accept any application in response to this RFA that is essentially
the same as one currently pending initial review, unless the
applicant withdraws the pending application.  The DRG will not accept
any application that is essentially the same as one already reviewed.
This does not preclude the submission of substantial revisions of
applications already reviewed, but such applications must include an
introduction addressing the previous critique.

REVIEW CONSIDERATIONS

Upon receipt, applications will be reviewed for completeness by the
DRG and responsiveness by the NHLBI.  Incomplete applications will be
returned to the applicant without further consideration.  If NHLBI
staff determine that the application is not responsive to the RFA, it
will be returned to the applicant without further consideration.

Applications that are complete and responsive to the RFA will be
evaluated for scientific and technical merit by an appropriate peer
review group convened by the NHLBI in accordance with the review
criteria stated below.  As part of the initial merit review, a
process (triage) may be used by the initial review group in which
applications will be determined to be competitive or non-competitive
based on their scientific merit relative to other applications
received in response to the RFA.  Applications judged to be
competitive will be discussed and be assigned a priority score.
Applications determined to be non-competitive will be withdrawn from
further consideration and the principal investigator/program director
and the official signing for the applicant organization will be
promptly notified.

Applications should be prepared so that they can be reviewed without
the necessity of interaction between the applicants and the
reviewers, since no site visit or reverse site visit will be part of
the technical review.

Review Criteria

The factors to be considered in the evaluation of scientific merit of
each application will be similar to those used in the review of
traditional research grant applications, including the novelty,
originality, and feasibility of the approach; the training,
experience and research competence of the investigator(s); the
adequacy of the experimental design; the suitability of the
facilities; the appropriateness of the requested budget to the work
proposed, and adequacy of plans to include both genders and
minorities and their subgroups as appropriate for the scientific
goals of the research.

AWARD CRITERIA

The anticipated date of award is July 1, 1995.  Funding decisions
will be made on the basis of scientific and technical merit as
determined by peer review, program needs and balance, and the
availability of funds.

In order to more evenly distribute administrative workload and reduce
the number of awards with July 1 or September 30 start dates, the
NHLBI will award ten months of time and money for the first competing
budget period of this project.  This action results in a project
period of 46 months rather than 48 months.  Investigators should plan
their research projects and budgets within these timeframes.

Awards in response to this RFA will be made to foreign institutions
only for research of very unusual merit, need, and promise, and in
accordance with PHS policy governing such awards.

INQUIRIES

Inquiries concerning this RFA are encouraged.  The opportunity to
clarify any issues or questions from potential applicants is welcome.

Direct inquiries regarding programmatic issues to:

Dr. Junius G. Adams, III
Division of Blood Diseases and Resources
National Heart, Lung, and Blood Institute
Federal Building, Room 508
7550 Wisconsin Avenue MSC 9090
Bethesda, MD  20892-9090
Telephone:  (301) 496-6931
FAX:  (301) 402-4843
Internet:  Junius_Adams@NIH.GOV

For fiscal and administrative matters, contact:

Ms. Jane R. Davis
Blood Division Grants Management Section
National Heart, Lung, and Blood Institute
Westwood Building, Room 4A11
Bethesda, MD  20892
Telephone:  (301) 594-7436
FAX:  (301) 594-7492
Internet:  Jane_Davis@NIH.GOV

AUTHORITY AND REGUALTIONS

The programs of the Division of Blood Diseases and Resources, NHLBI,
are described in the Catalog of Federal Domestic Assistance number
93.839.  Awards will be made under the authority of the Public Health
Service Act, Section 301 (42 USC 241) and administered under PHS
grants policies and Federal regulations, most specifically 42 CFR
Part 52 and 45 CFR Part 74.  This program is not subject to the
intergovernmental review requirements of Executive Order 12372, or to
Health Systems Agency Review.

.

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